| Common bean(Phaseolus vulgaris L.)is one of the main edible leguminous crops.Drought is the most important abiotic stress factor limiting the growth of common bean.Breeding drought-tolerant varieties is an important measure to reduce the adverse effects of drought on the growth of common bean.Drought tolerance of common bean is a complex trait controlled by multiple genes.Screening drought tolerance genes of common bean and analyzing their regulatory mechanisms are the basis of accurately breeding drought tolerance varieties.In the whole growth period of common bean,the seedling stage is the most sensitive to drought stress,which is the best time to screen the germplasm of drought tolerance and study the molecular mechanism of drought tolerance.In this study,60 representative common bean varieties were selected,and drought tolerance of common bean seedlings was evaluated by natural drought method in laboratory.Then,sequencing technology was used to analyze the complete transcripts of typical drought-tolerant varieties under drought stress(0h,24h,48h).The competitive endogenous RNA(ceRNA)regulatory network was constructed and drought-tolerant genes were screened out under drought stress.Finally,the drought tolerance gene SnRK2s were analyzed in detail.The main results are as follows:1.Identification of drought tolerance and screening of drought tolerance germplasm of common bean at seedling stageIn this study,60 representative common bean varieties were treated with drought indoors by natural drought method.12 indexes such as leaf conductivity,net photosynthetic rate,transpiration rate and photochemical quenching coefficient were measured in drought stress treatment group and control group,and drought tolerance coefficient of each individual index was calculated.The drought tolerance of 60 common beans was evaluated by seedling wilting grade and drought tolerance index weight(D value)of each variety.The results showed that F4321,F0117,F3370,F0518,F2179 and Nanyang Hei were identified as six drought-tolerant materials based on the classification of seedling wilting grade and D value.A mathematical model for drought tolerance evaluation of common bean seedling stage was established by stepwise multiple regression method:D=-0.577+0.157RPn+0.223RY(II)+0.112RNPQ+0.134RTr+0.172RFv/Fm+0.132RGs+0.086RCi-0.048REC+0.115RFv/F0+0.181Rq P+0.063RWC+0.006RWUE.The equation determined R~2=0.999,P=0.0001,and the average estimation accuracy was99.672%.According to the coefficient of determination,P value and average estimation accuracy of the equation,the equation could well estimate the evaluation value D of drought tolerance of common bean.2.Complete transcriptome analysis of common bean under drought stressIn this study,the typical drought-tolerant variety F3370 was selected as the material,and the dynamic transcriptomic changes of the first third leaf of common bean seedlings were detected by Illuminarna sequencing technology(RNA-Seq)under drought conditions(treated with 50 mmol/L mannitol for 24 and 48h).Under drought stress conditions for 24and 48 h,3 897 and 5 222 differentially expressed mRNAs,365 and 507 differentially expressed lncRNAs,27 and 74 differentially expressed miRNAs,4 and 5 differentially expressed circRNAs were identified,respectively.WRKY,MYB,NAC,bZIP,AP2/ERF,C2H2 zinc lipoprotein and other transcription factors related to drought tolerance were identified.Using these differentially expressed mRNAs,lncRNAs,circRNAs and miRNAs,the ceRNA network of common bean seedling was constructed under drought stress.GO enrichment analysis showed that these targets were related to water deprivation response,abscisic acid response,chlorophyll catabolism,polygalacturonase activity and other functions.KEGG analysis showed that these targets were involved in pentose and glucuronic acid conversion,plant MAPK signaling pathway,plant hormone signal transduction.In addition,to verify the accuracy of the sequencing results,8 differential mRNAs,4differential lncRNAs,4 differential miRNAs,and 4 differential circRNAs were randomly selected for qRT-PCR verification.The results showed that the trend of qRT-PCR was basically consistent with that of RNA-seq.3.Identification and expression analysis of SnRK2 gene in common bean under abiotic stressWith the help of common bean genome database information(https://v1.legume federation.org/data/v1/Phaseolus＿vulgaris/),based on the conserved domain of SnRK2protein sequence(Pfam:PF00069),the information of SnRK2 family genes related to drought tolerance in common bean was analyzed at the whole genome level by HMMER method.The results showed that there were 10 PvSnRK2 genes in the whole genome of common bean,which were named PvSnRK2.1～2.10.Phylogenetic analysis showed that these genes could be divided into four groups,PvSnRK2.1/2.8/2.10 belonged to groupⅠ,PvSnRK2.2/2.9 belonged to groupⅡ,PvSnRK2.5/2.6/2.7 belonged to groupⅢ,and PvSnRK2.3/2.4 belonged to groupⅣ.Stress response element(TC-rich repeats),drought induction(MBS)related MYB binding sites and low temperature response element(LTR)were identified in PvSnRK2s promoter region,indicating that SnRK2 gene family may be involved in low temperature and drought response.The typical drought-tolerant varieties F3370 and F4321 were treated with drought(mannitol),salt(NaCl)and low temperature(4℃),respectively,at the seedling stage of common bean.The expression profiles of 10 genes in leaves under different stress treatments were analyzed by qRT-PCR.The results showed that the expression of PvSnRK2.9 in F3370and F4321 reached the highest value at 48 h under drought stress,which were 8.31 times and3.58 times of the control,respectively.Suggesting that PvSnRK2.9 may play an important role in the response of common bean to drought stress.In addition,PvSnRK2.4 was activated in two common bean varieties under drought,salt and low temperature stresses,suggesting that PvSnRK2.4 may be one of the key regulatory gene in common bean response to osmotic and low temperature stress. |
PDF Full Text Request