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西瓜ClP5CS基因克隆及其功能研究
杨永超1,2, 王中元1, 杨小振,等1
1.西北农林科技大学 园艺学院;2.文山州农业科学院
摘要:
【目的】克隆西瓜自交系M08的ClP5CS基因(Cla006553Cla017928)cDNA序列全长,探究其在抵御干旱和盐胁迫中的作用。【方法】利用西瓜基因组信息,以M80幼叶为材料,克隆Cla006553Cla017928的编码序列,对其氨基酸序列进行生物信息学分析和同源分析;构建这2个基因的过表达载体转化农杆菌,采用浸花法浸染拟南芥,选取相应转基因T2拟南芥株系,检测其在脱落酸(ABA)、甘露醇、NaCl及干旱胁迫下的发芽率、根长、成活率及脯氨酸含量,研究Cla006553Cla017928对拟南芥抗逆性的影响。【结果】Cla006553编码序列长2 166 bp,编码721个氨基酸;Cla017928编码序列长2 145 bp,编码714个氨基酸。Cla006553Cla017928的核苷酸和氨基酸序列之间的相似性为分别为74.19%和77.12%,其氨基酸序列中含有ATP结合位点、脯氨酸反馈抑制位点、亮氨酸拉链、谷氨酰激酶结构域、NAD(P)H结合位点、亮氨酸结构域、谷氨酸半醛脱氢酶结构域。转Cla006553Cla017928基因的拟南芥植株在ABA、甘露醇、NaCl和干旱胁迫下的发芽率、根长、脯氨酸含量、成活率等均高于对照,具有较好的耐旱和耐盐能力。【结论】克隆了西瓜P5CS的2个编码基因Cla006553Cla017928,这2个基因均可增强转基因拟南芥植株的抗逆能力。
关键词:  西瓜  ClP5CS  拟南芥  渗透胁迫
DOI:
分类号:
基金项目:国家现代农业产业技术体系建设专项(CARS-26-18)
Cloning and functional characterization of ClP5CS genes in watermelon (Citrullus lanatus)
YANG Yongchao,WANG Zhongyuan,YANG Xiaozhen,et al
Abstract:
【Objective】This study cloned cDNA sequences of ClP5CS genes (Cla006553 and Cla017928) in the inbred watermelon accession M08 and verified their functions under drought and salt stress.【Method】Based on the information of watermelon genome,two full-length cDNAs of ClP5CS (Cla006553 and Cla017928) were isolated from M08 young leaves and analysis of bioinformatics and phylogenetic tree were carried out.Expression vectors carrying Cla006553 and Cla017928 were constructed and transformed into wild Arabidopsis via floral dip method.Independent lines of T2 were selected for further analysis based on their seed relative germination rates,length of roots,proline content and survival ratio of transgenic Arabidopsis plants under ABA,mannitol,salt and drought stress.【Result】The cDNAs of Cla006553 and Cla017928 were 2 166 bp and 2 145 bp in length,encoding 721 and 714 amino acid polypeptides,respectively.Sequence analysis showed that the two ClP5CS cDNAs shared an identity of 74.19%,and the amino acid similarity was 77.12%.The two ClP5CS proteins had conserved regions including ATP binding site,proline feed back inhibition site,leucine zipper,glu-5-kinase domain, NAD(P)H binding site,leucine domain and glutamic-γ-semialdehyde dehydrogenase domain.Cla006553 or Cla017928 transgenetic plants under ABA,mannitol,salt and drought stresses were better than control on seed relative germination rates,length of roots,proline content and survival ratio.【Conclusion】Cla006553 and Cla017928 were isolated from M08,and it was verified that they play important roles in increasing plants tolerance to osmotic stresses.
Key words:  watermelon  ClP5CS genes  Arabidopsis thaliana  osmotic stress