干旱诱导的甘蓝型油菜SSH文库及抗旱相关基因表达的分析  

Analysis of expression of drought associated genes and SSH library of Brassica napus under drought stress

作　　者：谢小玉[1] 侯爽 冉春燕[1] XIE Xiaoyu;HOU Shuang;RAN Chunyan(College of Agronomy and Biotechnology,Southwest University,Chongqing,Beibei 400715,China)

机构地区：[1]西南大学农学与生物科技学院,重庆北碚400715

出　　处：《湖南农业大学学报（自然科学版）》2020年第2期157-164,共8页Journal of Hunan Agricultural University(Natural Sciences)

基　　金：国家自然科学基金项目(31271673);公益性行业(农业)科研专项(201503127)。

摘　　要：从前期构建的干旱诱导的甘蓝型油菜SSH文库中随机挑取了646个阳性克隆,对其进行测序及生物信息学分析。结果表明:通过测序,获得639条高质量EST序列,186条单一序列(重叠群89条,单拷贝序列97条);比对分析发现,166条单一序列有同源序列;乙醛酸和二羧酸代谢、碳代谢、光合中的碳固定、氨基酸的生物合成、氮代谢等在植物抵抗干旱胁迫中发挥着重要的作用;MYB、NAC5、锌指蛋白等转录因子以及苹果酸脱氢酶、核酮糖1,5–二磷酸羧化酶/加氧酶、果糖–1,6–二磷酸醛缩酶、磷酸核酮糖激酶等与光合相关的基因以及P5CS基因、F–box蛋白等参与油菜干旱胁迫应答;单一序列涉及的功能中所占比例较大的是蛋白绑定和催化活性。以抗旱性较强的甘蓝型油菜Holiday为材料,在开花初期进行干旱处理,在处理的第1、3、5、7天(土壤含水量分别为60%、40%、30%、20%)进行叶片取样,采用实时荧光定量PCR,分析基因表达量,发现随机选取的文库中的3个基因(P5CSb、BnSOS2和CAM)在干旱胁迫下表达水平均上调,推测这3个基因在抵御干旱胁迫中发挥了重要作用。646 positive clones were randomly selected from the library of suppression subtractive hybridization(SSH) constructed at early stage. These clones were subject to sequencing and bioinformatics analysis. The results show that 639 high quality EST were got by sequencing, 186 unigenes was obtained, of which 89 contigs and 97 singletons were contained. Blast alignment with Nr database showed that 166 unigenes were of high similarity. KOBAS suggested that that glyoxylate and dicarboxylate metabolism, carbon metabolism, carbon fixation in photosynthetic organisms, biosynthesis of amino acids, nitrogen metabolism et al. played a key role in drought-resistant of rapeseed according to P-value. In the drought stress response, the genes of MYB family transcription factor, NAC5, zinc finger protein and other genes related to photosynthesis including malate dehydrogenase, ribulose 1,5-bisphosphate carboxylase/oxygenase, fructose 1,6-bisphosphate aldolase, phosphoribulokinase, carbonic anhydrase, aconitase like protein and delta 1-pyrroline-5-carboxylate synthetase A, F-box protein etc were inovled. The analysis of unigenes showed that a large share of function could be attributed to binding and catalytic activity. The high drought-tolerant rapeseed Holiday as experimental materials, were treated with drought stress during the flowering stage and with normal irrigation as the control group. We collected the samples at the 1st, 3rd, 5th and 7th day of drought stress(the soil water content was 60%, 40%, 30% and 20% respectively) and analyzed their expression values of P5CSb, BnSOS2 and CAM genes from screening the library by quantitative real time PCR reaction, their expression level were found up-regulated under drought stress in the leaves. This result suggested those genes may play an important role in the resistance of rape to drought stress.

关 键 词：甘蓝型油菜 干旱胁迫 cDNA文库分析 实时荧光定量PCR 

分 类 号：S565.4[农业科学—作物学] Q786[生物学—分子生物学]