qRT-PCR for Expression Analysis of Zma-miR397a and Zma-LAC Under Abiotic Stress
Info: 32802 words (131 pages) Dissertation
Published: 11th Dec 2019
Tags: BiologyEarth Studies
Abstract
Abiotic stresses are the environmental conditions that reduce growth, development and productivity in Z. mays. miR397a is a conserved miRNA which has potential functions in alleviating various abiotic stresses. Plant Laccase belong to the blue copper oxidase family and are involved in lignification. However, their physiological functions in plants are still not well understood. Little is known about the regulatory function of miR397a and Laccase in plant resistance to abiotic stresses. In a co-stress maize miRNA library constructed in our laboratory, these two genes showed antagonistic expression. In this research we used qRT-PCR for expression analysis of Zma-miR397a and Zma-LAC under abiotic stress. The results showed that Zma-miR397a and Zma-LAC were differentially expressed under drought, salt, alkali, low temperature, Abscisic acid (ABA), Salicylic acid and K+ deficiency treatments in shoots and roots of maize seedlings. Differential expression pattern of zma-miR397a and Zma-LAC was also recorded in different maize plant tissues. A 143bp pre-miR397a fragment flanked by 226bp upstream and 288bp downstream was amplified by PCR. The resultant sequence was analyzed by BlastN and showed 100% complementarity with the B73 pre-miR397a sequence from miRBase. The conserved region of Zma-LAC was amplified and sequenced. 3’ and 5’ RACE PCR were used to amplify the 3’ end and 5’ end respectively. The 1749bp Open Reading Frame (ORF) was amplified and sequenced. Amino acid sequence analysis confirmed that it is a Laccase due to its features. Zma-LAC was incorporated into the tobacco genome via Agrobacterium-mediated transformation. Transgenic plants were screened using PCR and semi-quantitative PCR. After salt stress and ABA treatment, transgenic tobacco plants showed root development as compared to wild-type plants. This study suggests that Laccase-mediated salt stress tolerance might be a common mechanism used by plants during growth under salinity stress.
Keywords: Z. mays, Zma-miR397a, Zma-LAC, qRT-PCR, tobacco, stress tolerance
Contents Page
Abstract……………………………………………………
Summary…………………………………………………..
1 Background………………………………………………..
1.1Effect of Salt stress on plants………………………………….
1.2 miRNA Literature Review…………………………………..
1.2.1 Plant microRNA Research Trend…………………………..
1.2.2 miR397 Family……………………………………..
1.2.3 miR397 involvement in plant growth and development………………
1.2.4 miR397 involvement in abiotic stress response…………………..
1.3 miR397a target gene(Laccase) Literature Review……………………..
1.3.1 Laccase gene research progress……………………………
1.3.2 miR397a-mediated regulation of Laccase ……………………..
1.4 Theoretical significance and application…………………………..
1.5 Aim and Objectives……………………………………….
2 miR397a and Laccase gene expression pattern analysis in maize…………………
2.1 Experimental materials …………………………………….
2.1.1 Plant materials and Abiotic stress treatments…………………….
2.1.2 Laboratory Reagents ………………………………….
2.1.3 Laboratory Equipment…………………………………
2.2 Methodology…………………………………………..
2.2.1 RNA extraction……………………………………..
2.2.2 qRT-PCR Primer design………………………………..
2.2.3 Total RNA extraction from Z. mays and first strand cDNA synthesis………
2.2.4 qRT-PCR…………………………………………
2.3 Results and analysis……………………………………….
2.4 Discussion…………………………………………….
2.5 Summary…………………………………………….
3. Cloning and characterization of pre-miR397a……………………………
3.1 Materials and methods……………………………………..
3.1.1 Plant material………………………………………
3.1.2 Laboratory Reagents…………………………………..
3.1.3 Laboratory Equipment…………………………………
3.2. Methodology…………………………………………..
3.2.1 Genomic DNA extraction……………………………….
3.2.2 Zma-pre-miR397a cloning……………………………….
3.2.3 Maize expression vector construction……………………………………………….
3.3. Results and analysis………………………………………
3.3.1 Cloning of pre-miR397a………………………………..
3.3.2 Zma-pre-miR397a sequence analysis…………………………
3.3.3 Plant expression vector construction…………………………
3.3.4 Transformation of A. tumefaciens with recombinant plasmid DNA……….
3.4 Discussion…………………………………………….
3.5 Summary…………………………………………….
4. Cloning and Bioinformatic analysis of Laccase…………………………..
4.1 Materials and methods……………………………………..
4.1.1 Plant materials………………………………………
4.1.2 Laboratory reagents…………………………………..
4.1.3 Laboratory instruments and equipment……………………….
4.2 Methodology…………………………………………..
4.2.1 Total maize RNA extraction………………………………
4.2.2 Cloning of the Laccase conserved region………………………………………..
4.2.3 Cloning of the Laccase gene 3’ end with 3’ RACE PCR……………..
4.2.4 Cloning of the Laccase gene 5’ end with 5’ RACE PCR……………..
4.2.5 Zma-LAC gene ORF cloning……………………………..
4.2.6 Laccase gene sequence Bioinformatic analysis…………………..
4.3 Results and analysis……………………………………….
4.3.1 Cloning of Laccase gene conserved region……………………..
4.3.2 Laccase gene 3’ end cloning………………………………
4.3.3 Laccase gene 5’ end cloning………………………………
4.3.4 Z. mays Laccase coding region determination……………………
4.3.5 Bioinformatic analysis of Laccase…………………………..
4.3.6 Plant expression vector construction…………………………
4.5 Summary……………………………………………..
5. Molecular and physiological screening of the transgenic tobacco plants…………….
5.1 Materials and methodology…………………………………..
5.1.1 Plant materials………………………………………
5.1.2 Laboratory reagents…………………………………..
5.1.3 Laboratory equipment………………………………….
5.2 Methodology…………………………………………..
5.2.1 Tobacco transformation…………………………………
5.2.2 Transgenic Tobacco plants molecular screening…………………..
5.2.3 Transgenic Tobacco plants physiological analysis…………………
5.3 Results and analysis………………………………………
5.3.1 Tobacco transformation…………………………………
5.3.2 Molecular screening of transgenic plants………………………
5.3.3 Physiological analysis tobacco transgenic plants………………….
5.4 Discussion…………………………………………….
5.5Summary………………………………………………………………………………… 69
Conclusion………………………………………………….
Future Pesperctive……………………………………………..
APPENDIX A……………………………………………….
APPENDIX B DNA Markers………………………………………
Acknowledgements…………………………………………….
Summary
Maize is an important cereal crop around the world. It is susceptible to abiotic stress hence this research is aimed at characterizing two genes which might be of help in stress resistance and increase in yield. The research will involve the use of qRT-PCR to observe the expression patterns of miR397a and Laccase in maize subjected to different abiotic stresses, phyto-hormones and nutrient deficiency and different tissues during growth. The complete sequence for Laccase will be cloned using PCR (conserved region), 3’RACE PCR (3’ end) and 5’RACE PCR (5’ end). PCR will be used to clone pre-miR397a. The sequences will undergo Bioinformatic analysis before being used to construct expression vectors. The recombinant expression vector harbouring the Laccase gene will be used to transform tobacco via the Agrobacterium-mediated method. Overexpression of this gene will open new insights on the possible role it might have during abiotic stress response.
1. Background
Abiotic stresses are the environmental conditions that reduce plant growth development and yield below expected levels such as drought, salt, alkali, frost among others. Drought is regarded as one of the major devastating environmental stresses which hampers plant productivity most compared to other stresses (Lambers et al., 2008). It drastically affects plant growth and development by reducing cell division and expansion rate, leaf size, stem and root elongation, plant water nutrient relation and water use efficiency (Li et al., 2009; Farooq et al., 2009). Salt stress is well known to affect all processes in a plant’s life cycle from germination to yield. It causes ion imbalance and hyperosmotic stress which affects the physiology of the plant (Zhu, 2001). These stresses influence various plant species to employ molecular, physiological and morphological ways of tolerance in order to survive (Cramer et al., 2011). The advancements in the field of molecular biology have revealed that different plant species are capable of responding to abiotic stresses at transcriptional and post transcriptional level.
- Effects of salt stress on plants growth
Salt stress is one of the major environmental factors that limit plant growth and productivity. It has adverse effect on germination, plant vigor and yield (Munns and Tester, 2008). High salinity levels affect plants in various ways such as ion toxicity, water stress, oxidative stress, nutritional disorders, membrane disorganization, reduction of cell division and expansion and alteration of metabolic processes (Munns, 2002; Zhu, 2007). During growth and development of plants, all major processes such as protein synthesis, photosynthesis, energy and lipid metabolism are affected (Parida and Das, 2005). After exposure to salt, plants experience water stress which in turn reduces leaf expansion. During long-term exposure to salt stress, plant undergo ionic stress which can lead to premature senescence of adult leaves in order to reduce the photosynthetic area available to support growth (Cramer and Nowak, 1992). Excess sodium and chloride have the potential to affect plant enzymatic activities resulting in reduced energy production levels (Munns, 2002). In maize, salt stress reduces shoot and root growth by suppressing leaf initiation and expansion, internode growth (Akram et al., 2010; Qu et al., 2012) and rate of cell elongation (Szalai and Janda, 2009) Several plant species have evolved several mechanisms either to exclude salt from their cells or to tolerate its presence inside the cells. Exploitation of genetic variations and generation of transgenic plants with novel genes or altered expression levels of the existing genes are ways in which plants can respond to salt stress.
Abscisic acid (ABA) plays an important role in response of plants to salt stress (Xiong et al., 2001). Salt stress signaling through ABA mediate the expression of late embryogenesis-abundant (LEA)-type genes including the dehydration-response element (DRE)/ C-repeat (CRT) class of stress-responsive genes Cor. Hence, the activation of LEA-type genes may actually represent damage repair pathways (Xiong et al., 2002). ABA dependent and independent signaling pathways mediate the salt and osmotic stress regulation of LEA gene expression. ABA-dependent and –independent transcription factors might also cross-talk to each other to amplify the response and improve salt stress tolerance.
Use of genetic manipulation approaches is required to unravel the mechanisms involved in salt response and to develop salt-tolerant plants which better cope with increasing soil salinity (Rajendran et al., 2009)
1.2 miRNA literature review
1.2.1 Plant miRNA research trend
miRNAs constitute a group of endogenous, single stranded, non-coding regulatory RNAs of about 19-24nt in length. They negatively regulate various developmental, molecular and physiological processes in plants. Plant miRNAs are involved in development, metabolic processes, hormone regulation, biotic and abiotic mediated stress response (Khraiwesh et al.,2012; Sunkar et al., 2012) and in the self-regulation of miRNA biogenesis. This is usually done by target mRNA slicing, cleavage (transcriptional) and translational inhibition. Target sites share high sequence complementarity to the respective miRNA and are often in coding regions though there are exceptions in the 3’ UTR (Bartel, 2004). By regulating their target proteins, miRNAs have been reported to be involved in diverse biological processes, including organ development, hormone signaling, defense against pathogens, and response to abiotic and biotic stresses. Important abiotic stresses in this regard include salinity, drought, cold, and heavy metals, nutrition, and other stresses. Almost all of these stress-induced miRNAs are evolutionarily conserved, which suggests that miRNAs-mediated regulatory mechanism may be evolutionarily conserved for corresponding environmental stresses in plants. However, the same miRNAs reported to respond abiotic stress in one certain species may not have the same function in other species (Sunkar and Zhu, 2004).
1.2.2 miR397a family
miR397 family is a conserved small family of miRNAs which constitutes of three members (miR397a, miR397b and miR397c). These members are isoforms of miR397 differing by 1-3 nucleotides in their mature miRNA sequences. miR397 in most of the plant species has the same mature miRNA sequence (UCAUUGAGUGCAGCGUUGAUG) which differ from the former by 2-3nt (Shen et al., 2010). Phylogenetic analysis of miR397 family was done by Shen et al., 2010 and it has been observed that 10 different plant species revealed several members that tend to be more closely related to those from other plant species than from the same plant species, implicating that evolution of the miR397 family occurred before the separation of these species. Known target genes of miR397 in Arabidopsis and Oryza sativa include tubulin beta-6, Laccase, Casein kinase and ß-fructofuranidase (Zhou et al., 2010; Jones-Rhoades and Bartel, 2004; Shen et al., 2010; Sunkar and Zhu, 2004). miR397 was seen to be involved in various abiotic stress responses, growth and development in various plants. miR397 involvement in plant growth and development
1.2.3 miR397 involvement in plant growth and development
miR397 has been observed to be involved during plant growth and development. Functional analysis has been conducted in several plant species to study miR397 interaction with its target gene (Laccases). miR397 contributes to growth and development and also has an effect on lignin synthesis and seed yield (Wang et al.,2014). In rice, miR397 has been involved in meristem development and embryogenesis (Luo et al.,2006). In another research, miR397 showed a high and specific expression in undifferentiated embryogenic calli, whereas displayed very low expression levels in differentiated calli and mature organs. miR397 target, Laccase, is principally involved in lignification and cell wall thickening during plant growth and development so this suggested that miR397 plays an important role in silencing of genes during meristematic development. The induction of miR397 in pro-embryogenic cells resulted in the antagonistic repression of Laccase which plays an important physiological role in maintaining embryonic cells in a thin wall and meristematic state. Low expression of miR397 allowed the accumulation of Laccase and this lead to the lignification of cell walls from the meristematic state to mature cell transition.
In Populus trichocarpa, Lu et al.,2011 showed that miR397 levels were high in phloem vessels, mature leaves and stem differentiating xylem as compared to young leaves, young stems and roots. Whereas in Arabidopsis, miR397(a/b) had significantly higher expression levels in the shoot apex, Inflorescence stems, seeds and whole seedlings as compared to rosette leaves or flowers. It was proposed that miR397 is specifically expressed in vascular seed plants so as to regulate lignin formation by cleaving Laccases (Wang et al., 2014). Hence it was suggested that miR397 might have evolved together with early vascular seed plants during their transition from aquatic to terrestrial life after it was detected in a number of plant species such as Norway spruce (Yakovlev et al.,2010), Loblolly pine (Lu et al., 2007; Quinn et al., 2014) but not in Selaginella (Banks et al., 2011) and Physcomitrella (Arazi et al., 2005; Talmor-Neiman et al.,2006).
It was also discovered that miR397 overexpression increases grain size and promotes panicle branching in rice plants and this is achieved by its ability to downregulate its target gene, Laccase (Zhang et al., 2013). Similarly, miR397b overexpressed Arabidopsis plants showed an increase in number of branches, siliques, seeds (size and number) (Wang et al., 2014). This unlike in Populus suggested that miRNA-mediated Laccase downregulation also contributes not only in vegetative growth but also reproductive and seed formation. Rice miR397 was characterized to be associated with the increase in grain size, weight and number which directly contributes to higher productivity. It was also suggested that rice miR397 was naturally highly expressed in rice seeds Chen et al., 2011. Such increase is likely due to the fact that rice miR397 represses the laccase gene (LAC), which is a key regulator of brassinosteroids signaling and is involved in various aspects of seed yield (Zhang et al., 2013). This was also supported by Peng et al., 2014 when he highlighted that miR397 is a potential grain filling regulator in rice due to its downregulating of Laccase and L-ascorbate oxidase target genes. Its target gene, Laccase 4 overexpression cause a reduction in inflorescent shoot number, seed (size and number). These findings suggest that miR397b is directly involved in growth and development in flowering plants.
To further elucidate the role played by miR397 in the post-transcriptional regulation of Laccases in plants, miR397 was overexpressed in Populus trichocarpa causing a reduction in Klason lignin. This showed a direct regulation system of miR397 mediated lignin biosynthesis during wood formation (Lu et al., 2013). Furthermore, miR397b overexpressing Arabidopsis plants showed a significant reduction in xylem tissues and lignin levels. miR397b target gene, Laccase 4, was overexpressed and it showed a significant increase in lignin content (Wang et al.,2014). These results suggested that miR397b and Laccase play a significant role in lignin content.
1.2.4 miR397 involvement in abiotic stress response
These stresses influence various plant species to employ molecular, physiological and morphological ways of tolerance in order to survive (Cramer et al., 2011). The advancements in the field of molecular biology have revealed that different plant species are capable of responding to abiotic stresses at transcriptional and post transcriptional level. miR397 is conserved in several plant species and is thought to be responsible for plant response to various stress conditions (Table 1). By observing miR397 expression patterns and overexpression, progress has been made towards increasing yield, resistance to abiotic stresses, nutrient homeostasis and hormone-mediated stress.
Table 1: miR397 involvement in abiotic stress response across various plant species
Species | Drought | Salt | Cold | Copper | H2O2 | Sulphate | Nitrogen | ABA |
A.thaliana | ↑ | ↑ | ↑ | ↑ | ↑ | ↑ | ↓ | ↑ |
P.trichocarpa | ↑ | ↑ | ↑ | ↑ | ↑ | ↑ | ||
B.distachyon |
|
↑ | ↑ | ↑ | ↑ | ↑ | ↑ | |
I.campanulata | ↓ | |||||||
J.pentantha | ↓ | |||||||
S.tuberosum | ↓ | ↓ | ||||||
S.melongena | ↑ | |||||||
R.raphanistrum | ↓ | |||||||
O.Sativa | ↓ | ↓ | ↓ | ↑ | ↑ | ↓ | ||
E.coracana | ↓ | |||||||
S.officinarum | ↑ | |||||||
P.virgatum | ↓ | ↓ | ||||||
P.coccineus | ↓ | |||||||
Z.mays | ↓ | ↓ | ↓ |
Drought is regarded as one of the major devastating environmental stresses which hampers plant productivity most compared to other stresses (Lambers et al., 2008). It drastically affects plant growth and development by reducing cell division and expansion rate, leaf size, stem and root elongation, plant water nutrient relation and water use efficiency (Li et al., 2009; Farooq et al., 2009). Plants in turn have methods of tolerating drought stress such as reduced water loss, enhanced water uptake and other biochemical and morphological changes (Bartel and Sunkar 2005).
miRNAs have been linked to drought stress since they are differentially expressed in this condition. Several investigations have indicated that plant miRNAs are involved in various important physiological processes such as seed germination, stem elongation and root proliferation during drought stress (Liu et at 2007; Sunkar et al., 2007). miR397a is also amongst the miRNAs involved during drought stress response in several species. In Arabidopsis thaliana (Liu et al., 2008; Sunkar and Zhu, 2004), B. distachyon and sugarcane (Gentile et al., 2015), miR397a was found to be induced during drought stress (Table 1). miR397 in Rice (Zhou et al., 2010), French bean (Nageshbabu et al., 2013), Finger millet (Nageshbabu et al., 2013b), I. campanulata, J. pentantha (Gorecha et al., 2013), S. tuberosum (Xie et al., 2010) and Switchgrass (Sun et al., 2012) was observed to be downregulated when subjected to drought stress. Laccase was reported to be the target gene in most of the species whereas ß-fructofuranidase was presumed to be rice miR397 target gene. This gene is mainly involved in sucrose and starch metabolism suggesting that miR397 plays an important role in CO2 fixation. This helps the plants to maintain a minimal rate of Carbon-Hydrogen manufacture to respond to drought stress (Zhao et al., 2010).
Salt stress is well known to affect all processes in a plant’s life cycle from germination to yield. It causes ion imbalance and hyperosmotic stress which affects the physiology of the plant (Zhu, 2001). In Arabidopsis thaliana (Liu et al., 2008; Sunkar and Zhu, 2004; Mangrauthia et al., 2013), Poplar (Zhang et al., 2009; Lu et al., 2008), B. distachyon (Lu et al., 2005, 2008), Eggplant (Zhuang et al., 2014) miR397 was found to be upregulated under salt stress but was observed to be downregulated in Zea mays (Ting and Su, 2015), Rice (Zhou et al., 2010; Khraiwesh et al., 2010; Sunkar et al., 2012; Zhao et al., 2007), Solanum tuberosum (Sun et al.,2012), Radish (Sun et al., 2015), Switch grass (Sun et al., 2012) and French bean (Nageshbabu et al., 2013) (Table 1). miR397 was differentially regulated in the above mentioned species thereby establishing its role in the adaptation of plants to salt stress. Its predicted target gene during the regulation was Laccase in several species which was reported to reduce root growth under dehydration (Cai et al., 2006).
Several plants, mostly those which are native to warm environments, show signs of injury when exposed to low temperatures. Signs and symptoms of cold stress induced injuries usually begin to show from approximately 48 to 96 hours although this differs from plant to plant. Cold stress also reduces reproductive development of plants. However, duration of exposure and the rate of decrease in temperature are also factors affecting the response from a plant. In Arabidopsis thaliana, during cold stress, miR397a and miR397b were upregulated (Liu et al., 2008; Sunkar and Zhu, 2004). Using wide transcriptome analysis, it was observed that miR397 was induced during exposure to cold stress conditions (Zhou, 2008). Laccases (AthLAC2, AthLAC4, AthLAC17) are known to be miR397 target genes and are involved in lignification. Kruszka et al., 2012 suggested that the downregulation of Laccase genes by miR397 might be part of a complex response which leads to the production of molecules which can be used for plant protection from cold. Using high-throughput sequencing experiments, miR397 was significantly upregulated and its target gene downregulated under cold stress in Brachypodium distachyon (Zhang et al., 2009) (Table 1). In 2014, Dong and Pei overexpressed miR397 in Arabidopsis thaliana and the resultant phenotype of plants showed resistance to chilling and also appeared larger in size as compared to the wild type plants after low temperature treatments. Cold regulating genes such as the Cold regulated (COR) gene family and CBF transcription factors were significantly higher in miR397-overexpressed transgenic plants thereby suggesting the contribution of miR397 in the cold signaling pathways.
It has been seen that several abiotic stresses trigger the accumulation of Reactive Oxygen Species which can cause oxidative damage to cells and sometimes cell death (Mittler, 2002). In rice, miR397 was found to be upregulated in response to oxidative stress and its target gene (validated to be Laccase) was downregulated. It was suggested that the downregulation of Laccase by miR397 minimizes the rate of biological processes which are nonessential and in turn save energy for tolerance and conserve copper which is used in several essential functions under severe stress (Li et al., 2010). In Arabidopsis thaliana (Sunkar and Zhu, 2004), Brachypodium distachyon (Ranocha et al., 2002; Zhang et al., 2009) and Poplar (Zhang et al., 2009; Lu et al., 2008), miR397 was also observed to be upregulated after exposure to oxidative stress and the target gene was downregulated in an effort to increase tolerance of the species.
ABA is a stress responsive plant phyto-hormone. It is well-known to regulate the expression of many genes in response to other environmental stresses. Under stress conditions it usually accumulates within the plant enhancing the production of Reactive Oxygen Species (ROS). Both ABA and ROS accumulation function as a stress signal that induces expression of antioxidant genes enhancing tolerance to the stress condition (Wei et al., 2009). Recent evidence indicates that miRNAs and ABA affect each other and that the expression levels of some miRNAs are regulated by ABA (Duan et al., 2016). miR397 has shown to be expressed in various plant species during ABA stress. In Arabidopsis, miR397 was found to be upregulated during ABA stress (Sunkar et al.,2012; Sunkar and Zhu, 2004; Bej and Basak, 2014).
miR397 plays a significant role in plant nutrient regulation. Copper is an essential micronutrient used by plants in various structural and biochemical ways such as cell wall metabolism, ethylene perception, oxidative stress protection and also as a co-factor protein involved in electron transfer reactions during photosynthesis and respiration (Burkhead et al., 2009). During copper deficiency in Arabidopsis, miR397 is significantly upregulated whereas its target genes (AthLAC2, AthLAC4 and AthLAC17) are downregulated. In rice, an increase in the level of miR397 was observed under copper deficiency conditions. It was proposed that as a way of adjusting the Copper homeostasis in the plant, miR397 targets and downregulates the expression of Copper Dismutases, Laccases and plantacyanin so as to conserve the available amounts of copper for more essential proteins such as cytochrome c and plastocyanin thereby facilitating the release of copper and restore it to optimum levels (Kruszka et al., 2012; Chun et al., 2005; Yamasaki et al.,2007; Abdel-Ghany and Pillon, 2008). Due to such biochemical changes, the plant begins to implement the morphological adaptations by utilising and recycling the copper from the senescing leaves.
Nitrogen deficiency in maize downregulated miR397 and antagonistically this triggered the upregulation of two Laccase gene family members. The two target genes in maize, the showed high sensitivity (Liang et al., 2012). The upregulation of the target genes caused various responses in plants such as their involvement in the reduction of root growth, energy metabolism and scavenging of oxidative species produced during stress (Xu et al., 2011).
miR397 possible plant nutrient crosstalk mediation was observed in nutrient homeostasis especially during Copper and Nitrogen deficiency as these two stresses proved to antagonistically influence each other thereby influencing the expression levels of miR397 during nutrient homeostasis (Liang et al., 2012). Low levels of a mineral element often affect absorption of other mineral elements and this phenomenon was observed when Copper starvation-induced miRNAs were suppressed by N starvation. miR397 was upregulated during low copper stress but further downregulated in low N conditions. It was suggested that the signaling molecules which are involved in Copper uptake were highly repressed in these low N conditions. It is highly imperative to look into the miR397 plant nutrient crosstalk mediation and further improve independent nutrient uptake during homeostasis.
In a study, 58 significant co-stress responsive maize miRNAs were identified and a library was constructed. In this research, miR397a was significantly downregulated seven fold under co-stress treatment (Ting and Su, 2015). These results coincided with those published on rice and finger millet. (Table 1.1)
1.3 miR397a target gene (Laccase) literature review
1.3.1 Laccase gene research progress
Laccases (EC 1.10.3.2) are copper-containing enzymes that have been studied in several organisms and have been proposed to participate in several physiological processes. It is conserved in plants, fungi, insects and bacteria. Laccases have been to be observed in several industrial applications, such as lignin degradation (Hood et al., 2003), soil detoxification (Sonoki et al., 2004) and polymerization (Bailey et al., 2004). Isolation and characterization of Laccases has been done in several species such as Arabidopsis thaliana (Berthet et al., 2011), loblolly pine (Sato et al.,2001), Poplar (Lu et al., 2013), Zea mays (Capparos-Ruiz et al., 2006), and cotton (Wang et al., 2004). Characterization has mainly been done in relation to lignification and enzyme activity. Seventeen genes that code for putative laccases have been found in the rice database (http://rgp.dna.affrc.go.jp/IRGSP/). Five laccases namely ZmLAC1 (AY897208) (Liang et al., 2006), ZmLAC2 (AM086214), ZmLAC3 (AM086215), ZmLAC4 (AM086216) and ZmLAC5 (AM086217) have been identified in a maize root library (Caparros-Ruiz et al., 2006). Forty-nine laccase gene models were identified in poplar (Lu et al., 2013) and seventeen were found in Arabidopsis (Berthet et al., 2011). Amongst the seventeen, eight were expressed in the stem and this suggested functional redundancy of laccases in lignin polymerization. In different studies, Laccases were reported to be induced following exposure to NaCl in tomato (Wei et al.,2000) and maize (Liang et al., 2006). This suggests and raised the possibility of laccase being involved in salt stress response.
1.3.2 miR397a-mediated regulation of Laccase
The Laccase gene family is regulated post-transcriptionally by multiple miRNAs. In Arabidopsis thaliana, four miRNAs were observed to target Laccase gene family members namely miR408, miR398, miR397 and miR857. The miR397 family was observed to target three Arabidopsis Laccase gene family members (AthLAC2, AthLAC4 and AthLAC17) (Salah et al., 2008; Li et al., 2010; Sunkar and Zhu, 2004) (Fig 1.1). Previous research has also confirmed the miR397a-mediated regulation of Laccase in various species such as rice (Zhang et al., 2013) and poplar (Lu et al., 2013).
Fig 1.1 Experimental validation of miR397a target genes in Arabidopsis thaliana. The target gene cleavage sites were determined by 5’RNA ligase-mediated RACE. (Salah et al., 2008)
1.4 Theoretical significance and application
The world population continues to grow and so is the food demand. Maize has been a crop with a high consumption the world over since it is used to alleviate famine and starvation in Africa as a whole and other developing countries around the world. Abiotic stresses conditions have caused serious yield losses and this has led to decrease in the food supply chain with the growing world population. This research on miR397a and Laccase genes highly promises understanding of the diversity and specificity of maize plant responses to different stresses. miR397a and Laccase gene cloning and characterization will help to provide a theoretical and functional understanding to further study on the role of these two genes in abiotic stress resistance.
1.5 Aim and Objectives
Zma-miR397a and its target gene, Zma-LAC were downregulated and upregulated respectively during exposure to co-stress treatment of drought, salt and alkali in our lab. This research will aim to reveal the expression and functional analysis of Zma-miR397a and Zma-LAC in maize and tobacco respectively. The expression pattern analysis of the two genes under various abiotic stresses and in different maize plant tissues will be investigated. In this study, we will also clone Zma-miR397a and Zma-LAC and further transform tobacco plants so as to lay a theoretical foundation of their function in alleviating abiotic stress.
2 miR397a and Laccase gene expression pattern analysis in maize
2.1 Experimental materials
2.1.1 Plant materials and Abiotic stress treatments
Zheng 58 Z. mays inbred were used in this research on abiotic stress. Maize seeds were placed in 65°C water in a 500ml flat-bottomed flask. The water temperature was allowed to drop o room temperature while stirring the flask. Seeds were transferred to a wet paper towel and allowed to germinate in a damp and dark place under room temperature. After approximately 5 days, the resultant germinated seedlings were transferred to silicon sand in small pots and were grown in an incubation chamber under the following conditions: temperature 24°C/20°C (day/night); photoperiod 16h/8h (day/night); humidity 60%. Further experiments were done after the seedlings had3 leaves and one heart approx. 7days. The maize plants were subjected to different abiotic stresses (NaCl, Drought, Cold, Na2CO3), phyto-hormones (Salicylic Acid, Abscisic Acid) and nutrient deficiency (Potassium deficiency). 25% PEG6000 was used to simulate drought conditions, 200mM NaCl for salt, 50mM Na2CO3 for alkali, 4°C for low temperature, 100mM ABA and 150mM Salicylic Acid(SA) for phyto-hormonal treatment. All treatments were done in full strength Hoagland nutrient solution. Potassium (K+) deficiency was done in Hoagland nutrient solution without the macronutrient K+. The duration of the treatment was 24h separated among 6 material collection time points (0h, 1h, 3h, 6h, 12h and 24h). All treatments were done in triplicate to ensure reliability. Roots and shoots were collected from 3 plants at every collection time point, mixed and frozen in liquid nitrogen prior to storage in a -80°C freezer for subsequent RNA extraction.
2.1.2 Laboratory Reagents used
Trizol (RNAiso Plus), PrimeScriptTM RT reagent kit with gDNA Eraser (Perfect for Real-Time), SYBR® Premix Ex Taq II (Tli RNaseH Plus) (2X), Easy Dilution (for Real-Time PCR) (Takara); RNase free water, PEG6000, NaCl, anhydrous Na2CO3, ABA (Sangon Biotech.Co., Ltd); SuperScriptIII reverse transcriptase (Invitrogen by ThermoFisher Scientific); Primer synthesis (Invitrogen Biotechnology by ThermoFisher Scientific Co., Ltd)
2.1.3 Laboratory Equipment
Laminar Air-Flow Cabinet(CA-1390-1), Phannaeia Biotech Image Master VDS, microwave oven (WP800TL23-K3) Galanz, China Co., Ltd, NanoDrop 2000c (Thermo Scientific Co., Ltd Shenyang, China), Thermal cycler TP600 (Takara)
2.2 Methodology
2.2.1 RNA extraction
- Collect 15-30mg of fresh plant material (roots or shoots) and immediately transfer into mortar, add liquid nitrogen, then crush with pestle to homogenize until powdery. Add the fine powder to a 1.5ml centrifuge tube with 1ml of RNAiso Plus and mix vigorously. Allow the mixture to homogenize for 5-10min at room temperature.
- To the solution from above, add 0.2ml chloroform per 1ml of RNAiso Plus. Cap the centrifuge tube and mix until solution becomes milky. Keep the solution at room temperature for 10minutes.
- Centrifuge at 12000X g for 15minutes at 4°C. The solution will separate into three layers; top liquid layer (contains RNA), semisolid middle layer (mostly DNA), and bottom organic solvent layer.
- Transfer the top liquid layer to new 1.5ml centrifuge tube without touching the middle layer.
- Add 1ml of isopropanol per 1ml of RNAiso Plus used for homogenization and mix well. Keep the mixture at room temperature for 10minutes.
- Centrifuge at 12000X g for 15minutes at 4°C to precipitate the RNA
- Carefully remove the supernatant. Add 1ml of 75% cold ethanol and vortex. Centrifuge the solution at 8000X g for 5minutes at 4°C and discard the supernatant (Repeat this process twice)
- Dry the precipitate by leaving the tube open for several minutes and add 20µl RNase free water
- Analysis by absorbance is done by measuring 1ml of dissolved RNA using NanoDrop 2000 and further diluting the stock to a concentration of 500ng/µl.
- Analysis of RNA purity is done by agarose gel electrophoresis
2.2.2 qRT-PCR Primer design
Primers were designed according to the existing sequence of Zea Mays B73 which was found in NCBI Database. The target gene (Laccase) primers, LAC-F and LAC-R for Real-Time quantitative PCR(qPCR) were designed using IDT Primer Quest Tool. Stem loop RT-qPCR primers containing a specific extension at the 3’ end that is the reverse complement of the last seven nucleotides at the 3’ end of miR397a products. The forward primer miR397a-F was specific to the miR397a mature sequence. The universal reverse primer, miRNA-R is specific to the 5’ end of the stem-loop RT primer. miR172 and U6 were used as endogenous normalized genes. Primer sequences are shown in a table below.
Primer name | Primer Sequence(5′ -3′) |
LAC-F | GGTTTGGTGTTATGCTTCCATTTAG |
LAC-R | CTAGTAGCATCAACGGACCTTC |
miR397a-F | TCATTGGTGTTATGCTTCCATTTAG |
miR397a-RT | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCGGCA |
miR172-F | GCCGGCGAATCTTGATGATGC |
miR172-RT | GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTGCAGC |
miRNA-R | GTGCAGGGTCCGAGGTATTC |
ZmU6-F | TTGGAACGATACAGAGAAGATTAGC |
ZmU6-R | AATTTGGACCATTTCTCGATTTGTG |
2.2.3 Total RNA extraction from Z. mays and first strand cDNA synthesis
Total RNA from abiotic stress treated Z. mays Zheng58 was extracted using the RNAiso reagent protocol by the manufacturer. Quantity and quality of RNA was determined by Nano Drop 2000 and concentration was set to 500ng/µl. The purity analysis of RNA was done by agarose gel electrophoresis.
- PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) was used for reverse transcription of target gene cDNA. The following protocol was used:
- Genomic DNA elimination reaction (reaction prepared on ice)
5X gDNA Eraser Buffer 2µl
gDNA Eraser 1µl
Total RNA 2µl
RNase Free dH2O Up to 10µl
Reaction conditions for themocycler: 42℃ 2 min
- Reverse-transcription reaction (reaction prepared on ice)
Reaction solution from step 1) 10µl
5X PrimeScript Buffer 2 (for Real Time) 4µl
PrimeScript RT Enzyme Mix I 1µl
Master mix
RT Primer Mix 4µl
RNase Free dH2O Up to 20µl
Reaction conditions for thermocycler: 37℃ 15 min
85℃ 5 s
4℃ ∞
- SuperScript ™ III reverse transcription reaction kit was used for miRNA reverse transcription. The following protocol was used:
- Total RNA 8μl
2.5mM dNTP Mix 4μl
Gene-specific primer(2 pmol/μL) 1μl
Reaction conditions for thermocycler: 65℃ 5min
Place on ice: 1min
- Reaction solution from step 1)
5×First-stand Buffer 4μl
0.1 DTT 1μl
RNase OUT 1μl
SuperScript™ III RT 1μl
Reaction conditions for thermocycler: 55℃ 60min
70℃ 15min
4℃ ∞
2.2.4 qRT-PCR
real-time PCR using SYBR® Premix Ex Taq II (Tli RNaseH Plus) kit was used as described by the manufacturer. Target gene and miRNA cDNA was diluted 5-fold and 10-fold respectively after standard curve analysis. The qRT-PCR reaction was prepared as follows:
SYBR Premix Ex Taq II (Tli RNaseH Plus) (2X) 12.5 μl
PCR Forward Primer (10 μM) 1.0 μl
PCR Reverse Primer (10 μM) 1.0 μl
RT reaction solution (cDNA)*2 2.0 μl
Sterilized purified water 8.5 μl
qRT-PCR optimal reaction conditions: Hold (initial denaturation)
Cycle:1
95℃ for 20 sec
2 Step PCR
Cycle:40
95℃ for 5 sec
60℃ for 20 sec
Dissociation
The ddCT method was used to analyse relative gene expression. All reactions were done in triplicate (Biological and technical)
2.3 Results and analysis
In order to study the expression pattern of miR397a and its target gene, Laccase, in different abiotic stresses, real-time PCR was performed on the leaves and roots of the maize plants at different time points (0h, 1h, 3h, 6h, 12h, 24h). Abiotic stress treatments which were investigated include PEG 27% (Drought), NaCl 200mM (Salt), Na2CO3 50mM (Alkali), ABA 100µM (Hormone), Salicylic acid 50µM (Hormone), Cold 4˚C and Potassium (K+) deficiency. Root samples and shoot samples (stem and leaf) underwent through RNA extraction procedures. The RNA was tested for quality and reverse transcribed to cDNA prior to qualitative real-time PCR. The relative expression of miR397a and Laccase was measured and analyzed. Following similar protocols as above, the miR397a and Laccase gene expression were analyzed in different tissues during maize growth from juvenile to reproductive stage. Particular tissues which were investigated include leaf, root, stem, silk, tassel and immature seed and these were collected at different stages of maize growth. This was also done to see the relative abundance of the two different genes in these different tissues during growth.
2.3.1 miR397a and Laccase gene expression during abiotic stress treatment
1) miR397a and Laccase relative expression during Drought stress treatment
RNA extraction and quality control
RNA extraction was performed using the trizol method and its concentration was measured using the Nanodrop2000c. Total RNA concentration by A260 was maintained at 500ng/µl. For purity, A260/A280 ratio was kept at 1.8-2.0 where as A260/A230 was 2.0. Integrity of the RNA was detected by agarose gel electrophoresis, which showed clear 28S and 18S ribosomal RNA (rRNA) bands, no nucleic acid degradation or genomic DNA. This analysis showed that RNA integrity was standard and could undergo reverse transcription prior to qRT-PCR (Fig 2.1).
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