| Adversity stresses such as drought,heat,salinity,UV light and frost are the global threat,resulting in crop losses and environmental deterioration.The xerophytes living in desert steppes have evolved specialized strategies to survive in harsh environments.Understanding the molecular mechanisms,including identifying key stress-resistance genes from xerophytes,is important for our fundamental knowledge of plant stress adaptation.It also allows for genetic improvement of fine forages likely Medicago sativa,which have been cultivated under favourable farming regimes for a long period and thus possess very limited natural stress resistance.Zygophyllum xanthoxylum,widely distributed in desert regions of north western China,has evolved a series of adaptation strategies,enriches with stress-resistant genes,is suitable for genetic improvement of alfalfa.In our previous investigation,Na+/H+ antiporter(Zx NHX)and H+-PPase(Zx VP)gene responsible for cell Na+ compartmentalization,were used to improve the salt,barren and drought tolerance of alfalfa,which was mainly based on the ability of osmotic adjustment of Z.xanthoxylum.However,making use of its well-developed and thick surface cuticle gene to improve alfalfa has not been reported.In this study,based on the cloning of the cuticle lipid transporter gene ZxABCG11 and the epidermis-specific promoters AtCER6 and MtML1 in our previous work,in order to obtain transgenic alfalfa expressing ZxABCG11,and then conduct hybridization with the transgenic alfalfa co-expressing ZxNHX-ZxVP1-1,finally harvest the safety transgenic alfalfa co-expressing ZxABCG11,ZxNHX and ZxVP1-1 genes which have strong osmotic adjustment ability and cuticular coat withstanding the drought,salinity,UV and heat,the following experiments would be done: the plant expression vectors construction,ZxABCG11 functional verification in drought resistance,genetic transformation to alfalfa.The main results are as follows:1.The fusion gene ZxABCG11-FLAG were combined by PCR;Using pmi as a safe selectable marker gene,promoter AtCER6 and MtML1 as the epidermis promoters,two plant expression vectors were constructed through double digestion method named as AtCER6 :: ZxABCG11-FLAG :: 35 s :: pmi and MtML1 :: ZxABCG11-FLAG :: 35 s :: pmi.2.Using AtCER6 :: ZxABCG11-FLAG :: 35 s :: pmi and MtML1 :: ZxABCG11-FLAG :: 35 s :: pmi expression vectors by Agrobacterium-mediated genetic transformation of floral dip in Arabidopsis,we got transgenic plants.The ectopic expression of ZxABCG11 enhanced drought tolerance in transgenic Arabidopsis.The biomass,net photosynthetic rate(Pn),water use efficiency(WUE),chlorophyll content,leaf relative water content(RWC)of the transgenic lines AAP1,AAP2,MAP1,MAP2 were higher than wild type.In contrast,the rate of water loss,transpiration rate and leaf membrane permeability of leaves were lower than wild type.At the same time,the survival rates of MAP1,MAP2,AAP1 and AAP2 were 27.3%(12/44),28.9%(13/45),66.7%(30/45)and 33.3%(15/45)respectively,while wild type(WT)all died(0/45).In addition,we found that the transgenic lines preliminarily focused on vegetative growth and later promoted reproductive growth.These above results show that ZxABCG11 gene is an excellent drought resistance gene,and it is also proved that the constructed vector is active and can be used for the genetic improvement of alfalfa.3.Based on our previous transformation system,we modified D-mannose selection procedures: D-mannose resistant somatic embryos and plants were selected by 25/5 g·L-1 and 15/15 g·L-1(D-mannose/sucrose)after 14 d when somatic embryos and roots were induced,respectively.Finally,This research firstly transformed a cuticle lipid transporter gene ZxABCG11 from Z.xanthoxylum into alfalfa cultivar Xinjiang Daye and obtained 59 D-MannoseR lines.In a word,this study will contribute to breaking the bottleneck of stress-resistance gene scarcity in genetic improvement of cultivated forages,and establish the foundation for harnessing desert and saline land. |
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