Application Of SNAC1 Gene In Cotton And Analysis Of Cotton Genomic DNA Methylation Under Salt Stress

Plant’s adaptability to abiotic stresses such as drought,cold and salinity plays an important role in plant’s yield and quality.Drought and salinity stresses are the most common form of adversity,which are the limiting factors for agricultural development in many areas.Therefore,it is important to improve crop’s tolerance to stresses by genetic engineering.Plant genomic DNA was always methylated under stresses and studying the level and pattern of genomic DNA methylation under stresses will help to identify the mechanisms on stress resistance.Cotton is one of the most important economic crops in the world,and genetic engineering played an important role in increasing cotton’s yield,enhancing stress resistance and improving cotton fiber quality.The objective of this study was to gain transgenic cotton plants for genetic improvement,SNAC1 gene,a transcriptional factor regulating stress response,which was transformed into cotton and the analysis and identification of stress resistance were carried out on transgenic cotton plants systematically.The availability of SNAC1 gene was indentified preliminarily in genetic improvement and SNAC1 gene as a selection marker gene for genetic transformation of cotton was studied also.In addition,the cotton strain of YZ1 was used to study the level and pattern of DNA methylation in cotton roots under salt stress.The following results were obtained:1.SNAC1 gene in rice can enhance drought- and salt-tolerance,and then was transformed into Gossypium hirsutum YZ1 by Agrobacterium -mediated genetic transformation in this study.PCR detection and RT-PCR analysis indicated SNAC1 gene had been integrated into the transgenic cotton genome and at the same time regenerated plants of 9 clones were gained totally.The field surveys showed that T₁ generation of transgenic plants did not vary in many agronomic traits such as yield,compared to non-transgenic plants.The determination of rate of water loss of excised-leaves of cotton demonstrated that the RWL（rate of water loss） of T₁ generationof transgenic plants was 25%,and was lower than that（30.4%） of the non-transgenic plants.Then the observation of SEM（scanning electron microscope） also suggested the ratio of stomatal closure in the leaves of transgenic plants was 71.8%and was higher than that（51.5%） of non-transgenic plants.The results of salt-tolerance identification by water culture showed that RWC （Relative water content） of leaves of T₂ generation of transgenic seedlings was 72%under stress of 200 mmol/L NaCl after 5 d and that of non-transgenic cotton’s leaves was 61%. What is more,the leaves of non-transgenic plants withered and broken off earlier than that of transgenic plants.In addion,the RGR（Relative growth rate） of transgenic plant seedlings were 7.9%and 109.5%respectively during salt stress and sress revovery,and the RGR of non-transgenic plants were only-7.4%and 53.1%respectively.Under the 150 mmol/L NaCl stess transgenic embryogenic calli can continue to grow,but non-transgenic embryogenic calli lost water and died.The results of qRT-PCR analysis suggested that in T₁ generation of transgenic positive and negative plants SNAC1 gene could regulate expressions of some genes in cotton,for example expressions of G2（NAC family protein） and G3（NAM-like protein） genes were up-regulated and expressions of G5（GSA-AM2）, G11（SAMS） and AF38（ABF2） genes were down-regulated.2.In this study,employing SNAC1 gene was selected as a marker gene and NaCl as a selection agent,SNAC1（Stress-induced NAC 1） and GUS genes were introduced into cotton genome via Agrobacterium-mediated transformation,and the feasibility of NaCl as a selection agent for the genetic transformation of cotton was researched.Finally transgenic embryonic calli were gained after the infection with Agrobacterium,co-culture and selection culture of NaCl.Transgenic calli were confirmed by PCR analysis and positive rate of transgenic calli was 58%,and at the same time expression of the GUS gene was showed with GUS staining.Furthermore the concentration of NaCl and method for employing NaCl as a selection agent had been studied.The hypocotyls were co-cultured for 3～4 d after Agrobaterium infection.The reasonable selection concentration of NaCl should be 1.1%～1.5%（W/V） and the start concentration of NaCl in the callus induction medium should be lower.Then the concentration was increased as cotton calli proliferated.Because NaCl is not beneficial for embryo differentiation and development,NaCl should be removed from the culture medium to promote embryonic calli differentiation after calli were subcultured for 3～4 times.3.In this study the growth of cotton seedlings and the level and patterns of DNA methylation in the roots of cotton were investigated uder salt stress.The results showed that 100 mmol/L NaCl obviously promoted plant height and root length of cotton seedlings,but 200 mmol/L NaCl significantly inhibited the growth of cotton seedlings; 100～200 mmol/L NaCl inhibited the numbers of lateral root considerably.The analysis of MSAP showed that the level of global DNA methylation in the roots of cotton seedlings was 41.2%,38.1%,35.2%and 34.5%respectively under the stresses of 0,100,150,200 mmol/L NaCl;there was a significantly negative correlation（r=-0.986） between NaCl concentrations and the level of DNA methylation in the roots of cotton seedlings. Compared to the control（0 mmol/L NaCl） the methylation ratio of root genomic DNA were 6.4%,7.6%and 11.3%respectively,and the demethylation ratio of root genomic DNA were 12.7%,11.1%and 8.2%respectively under the stresses of 100,150 and 200 mmol/L NaCl.In addition,the analysis of sequences suggested that parts of MSAP fragments were homologous to functional genes.The results of RT-PCR showed that M3 fragment homologous to gypsy retrotransposon reverse transcriptase gene of Gossypium barbadense and M2 fragment homologous to Gossypium hirsutum cDNA GH_TMO were expressed in the roots of cotton seedlings under 200 mmol/L NaCl stress,but were not expressed under 0 mmol/L NaCl stress.