Germplasm Evaluation, CDNA Library Construction, Genes Cloning And Identification Of Drought Resistance In Cleistogenes Songorica

Native plants are a potentially rich resource for discovery of novel genes and gene variants.Functional genomics and genes discovery specifically targeting native plants can provide a view on the mechenism of abiotic stress tolerance and derive improved crops and forages with enhanced tolerance to abiotic.Cleistogenes songorica is a xerophytic perennial grass.Although it had very important value,the value of drought tolerance and as a potential plant for regeneration of desert-steppe areas have only recently been recognized.In this project,native C.songorica germplasm were collected for drought tolerance mechanism understanding and drought responsive genes discovery. Expressed sequence tags(ESTs)were generated from cDNA libraries prepared from water stressed leaf and root.We analyzed the expression level of drought reponsive genes in C.songorica and after transformed in arabidopsis,which will provide basic information of those genes for genetic improved forage grasses.The progress was as followings.In this study,16 native C.songorica germplasm were collected in China.Genetic diversity of the C.songorica populations were studied at RAPD levels.A total of 192 individuals of 16 populations were analyzed using 7 primers,producing 52 scorable bands,23 of which were polymorphic.An analysis of molecular variance(AMOVA) revealed a much larger genetic variation among populations(53.19%)than between them (46.81%).A significant proportion was attributable to differences among populations(P＜0.001,tested using a 1000 permutations),but not within populations.The UPGMA Cluster analysis showed two groups according to community type and altitude.The first group was including 10 populations of pop1～pop5,pop7 and pop13～pop16,while 6 populations of pop6,pop8～pop12 were included in second group.Three highly divergent populations of CS01,CS09 and CS15 were imposed drought stress by withholding irrigation and then rewatering.Leaf water content, photosynthesis,chlorophyll florescence and pigment content were studied for our understanding of the mechanisms involved in plant response and tolerance to water stress. During the withholding irrigation period,leaf relative water content is 75%,and leaf water potential is -3.2 Mpa,so C.songorica can be seen as a drought tolerant plant with high level of leaf water potential.Drought induced a decline of photosynthesis and pigment content rather than a direct damage to PSâ…¡.Only Fv/Fm was observed to decline during the drought stress experiment,other PSâ…¡photochemistry parameters were not affected.Different changes existed between photosynthesis and intercellular CO₂ concentration,which tell us that the decline of the photosynthesis in C.songorica attribute to the decline of assimilation capacity of mesophyll cells.Using SMART method,4 cDNA libraries were constructed from leaf and root of C. songorica with light and severe drought stress.The recombination rate of Cs01L,Cs02R, Cs03L and Cs04 library was 78.6%,100%,78.6%and 71.4%respectively,with the insert fragment size of 0.2～2.8kb.Randomly selected cloned were used to establish a genomic resouce of expressed sequence tags(ESTs).A total of 5664 sequence were generated with 3579 of them being of suffient quality for further analysis.This produced a database of 2,191,166 bases of C.songorica sequence.The 3579 ESTs were assembled into 1499 contigs with 805 singleton unigenes and 694 multi-memeber unigenes using the Phrap program.Gene Ontology(GO)functional annotation was assigned to the unigenes by sequence similarity to entries within Uniprot using BLAST,with 60%(899) of the unigenes annoted with GO term.Categories were assigned on the basis of molecular function,biological process and cellular component with 22%,46%and 32% respectively.63 unigenes were annoted as stress responsive genes,including 25 drought responsive unigenes.Using SSRPrimer program,we searched for potential microsatellite (SSR)sequences in the 1499 candidate gene set.In total,161 SSR were identified from C. songorica sequence databases.Using RACE method,10 full length of drought responsive genes were cloned. GeNorm software was used to analyze gene expression stability.The expression stability of 7 house keeping genes were CsEIF5₂＞CsEIF5₁＞CsGAPDH＞CsActin-11＞CsUBQ＞CsTUA2＞CsHistone.GeNorm analysis indicated that GAPDH,CsEIF5₁ and CsEIF5₂ are the most stable genes,Hence,after calculation of a nomalization factor from 3 most stable genes,the normalized expression levels can be calculated by dividing the raw quantities for each sample by the the appropriate normalization factor.By screening the expression level of 22 drought response genes using RT—PCR and relative quantification of qPCR,8 genes were observed to induce by drought stress and differentially expressed under different stress treatment.To understand the mechanism of expression and regulation of 7 genes,absolute quantification was employed to analyze its expression under diverse tissues and stress treatment(0d,4d,6d,8d,10d after withholding watering and 1d,4d after rewatering).CsALDH gene was expressed about 17 folds in drought stressed root tissue than in the controls.CsLEA2 gene was only observed to express in drought stressed plants.It began to express slightly in root tissue under 4 days of withholding watering,then increased after 6 days and got the maximum expression after 8 days and 10 days of withholding watering.The expression level was decreased significantly after 1 day of rewatering and no expression was detected after 4 days ofrewatering.Using Gateway cloning technology,CsALDH and CsLEA2 genes were constructed into plant expression vectors contaning constitutive expression promotor CaMV 35S or stress induced promotor rd29A,which were thansferred to Arabidopsis plant.