生菜Ls PHYB可变剪接体的克隆与高温诱导表达模式  

Cloning of Alternative Splice Variants of LsPHYB in Lettuce and Its Expression Patterns Under Heat Stress

作　　者：隋心意 赵小刚 陈鹏宇 李亚灵[1] 温祥珍[1] SUI XinYi;ZHAO XiaoGang;CHEN PengYu;LI YaLing;WEN XiangZhen(Horticulture College,Shanxi Agricultural University,Jinzhong 030801,Shanxi)

机构地区：[1]山西农业大学园艺学院,山西晋中030801

出　　处：《中国农业科学》2022年第9期1822-1830,共9页Scientia Agricultura Sinica

基　　金：山西省重点研发计划(201803D221001-1)。

摘　　要：【目的】光敏色素B(phytochrome,PHYB)是光和温度的受体。通过克隆光敏色素B基因(PHYB)可变剪接体并分析其在高温诱导下的表达模式,探究LsPHYB可变剪接体在生菜响应环境高温中的生物学功能,为培育耐热性生菜提供理论依据。【方法】采用生物信息学方法在生菜的基因组数据库搜索获得LsPHYB的cDNA序列的相关信息;对克隆得到的3个可变剪接体LsPHYB1、LsPHYB2和LsPHYB3进行多序列比对、可变剪接方式分析及系统进化树分析;通过在线软件预测PHYB1、PHYB2和PHYB3蛋白分子量、等电点和亲水性、疏水性等蛋白质理化性质,并通过生物信息学软件预测三者的二级结构、三级结构和保守结构域;采用荧光定量PCR(qRT-PCR)检测PHYB1、PHYB2和PHYB3在高温处理后的表达特征。【结果】克隆获得的生菜LsPHYB的3个可变剪接体LsPHYB1、LsPHYB2和LsPHYB3的CDS长度分别为3509、3877和2690 bp,编码氨基酸长度分别为1094、960和853 aa。其中LsPHYB1发生可变3′端位点和外显子跳跃类型可变剪接,LsPHYB2发生选择性保留polyA尾和内含子保留型可变剪接,LsPHYB3发生外显子跳跃类型可变剪接。保守结构域分析表明PHYB2的N端缺少PAS和PHY功能域;PHYB3的N端缺少PAS和PHY功能域,C端缺少HisKA功能域;系统进化树分析表明,3个可变剪接体聚为一支。qRT-PCR分析表明在高温处理第1天,LsPHYB3的表达量最高;在高温处理第5—9天,LsPHYB2的表达量高于LsPHYB1和LsPHYB3;在高温处理第11天,LsPHYB1的表达量高于LsPHYB2和LsPHYB3,处理11 d内三者表达量达到峰值的时间不同。【结论】高温下生菜LsPHYB的转录本存在3个可变剪接体LsPHYB1、LsPHYB2和LsPHYB3。LsPHYB3在高温处理前期高表达,LsPHYB2、LsPHYB1分别在高温处理中期、后期高表达,推测生菜3个LsPHYB可变剪接体在抗高温胁迫中发挥不同的作用。【Objective】Phytochrome B(PHYB) is a receptor for both light and temperature. In this study, the biological functions of alternative splice variants of Ls PHYB in lettuce in response to high environmental temperatures were investigated through cloning them and analyzing their expression patterns, so as to provide a theoretical basis for breeding heat-tolerant lettuce.【Method】The c DNA sequences of Ls PHYB were searched from the genomic database of lettuce by bioinformatics method. The cloned LsPHYB1,Ls PHYB2 and Ls PHYB3 were subjected to multi-sequence alignment, alternative splice variants form analysis and phylogenetic analysis. Protein properties, such as molecular weight, theoretical isoelectric point, hydrophilicity and hydrophobicity, were predicted by online software. Secondary structure, tertiary structure and conserved domains were analyzed by bioinformatics software. Three alternative splice variants were characterized for expression after high temperature treatment by RT-PCR. 【Result】There were three alternative splice variants of LsPHYB obtained by cloning, namely LsPHYB1, LsPHYB2 and LsPHYB3, with their CDS lengths of 3 509, 3 877 and 2 690 bp, which encoded 1 094, 960 and 853 amino acids, respectively. Alternative splice forms of LsPHYB1 were alternative 3′ splice site and skipped exon. Alternative splice forms of Ls PHYB2 were alternative polyA and retain intron. An alternative splice form of LsPHYB3 was skipped exon. Conserved structural domain analysis showed that the N-terminal of PHYB2lacked the PAS and PHY domains. The N-terminal of PHYB3 lacked the PAS and PHY domains, and its C-terminal lacked the HisKA domain. Phylogenetic analysis showed that three alternative splice variants were clustered into a clade. q RT-PCR analysis showed that the expression of Ls PHYB3 was the highest at the first day of high temperature treatment;Ls PHYB2 had higher expression than Ls PHYB1 and Ls PHYB3 at days 5-9 of high temperature treatment;at day 11 of high temperature treatment, the expression

关 键 词：生菜 LsPHYB 可变剪接体 高温胁迫 响应 

分 类 号：S636.2[农业科学—蔬菜学]