山黧豆LsSULTR基因的克隆与表达分析
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CHEN Hong, ZENG Peng, XU Quanle* (西北农林科技大学 生命科学学院陕西杨陵 712100) 
基金项目:陕西省自然科学基金(2020JM 167);
中文摘要:为研究山黧豆(Lathyrus sativus)硫酸盐转运蛋白(sulfate transporter,SULTR)与内源活性物质β N 草酰 L α,β 二氨基丙酸(β N oxalyl L α,β diaminopropionic acid,β ODAP)含量之间的关系,该研究采用序列比对的方法,从山黧豆转录组数据(SRP145030)中查找SULTR基因序列,并进行生物信息学分析;采用PCR方法克隆基因全长序列并进行组织特异性表达分析,以筛选与β ODAP含量相关的SULTR基因。结果表明:(1)经序列比对共查找到13条SULTR基因序列,其编码蛋白分别隶属于SULTR Ⅰ-Ⅳ。其中LsSULTR3;3和LsSULTR3;5具有SULTR蛋白家族保守的STAS结构域 (PF01740) 和Sulfate_transp结构域(PF00916),且二者活性受到转录因子MYB和激素响应等多个顺式作用元件的共同调节,并受蛋白水平互作及磷酸化等翻译后修饰调控。(2)成功获得LsSULTR3;3和LsSULTR3;5基因全长cDNA分别为1 962 bp和1 923 bp,分别编码653和640个氨基酸。(3)半定量RT PCR分析显示,LsSULTR3;3在山黧豆主根、成熟叶、茎、花、盛荚初期(S2)种子和鼓粒满期(S6)种子中表达,并在茎中的表达量较高;LsSULTR3;5在山黧豆主根、侧根、花、S2时期种子中均有表达,且花中的表达量最为显著。该研究结果为进一步研究山黧豆中硫酸盐的吸收、转运及同化、β ODAP的生物合成途径等奠定了基础。
中文关键词:山黧豆  硫酸盐转运蛋白  β ODAP  基因克隆  蛋白活性调控
 
Cloning and Expression Analysis of LsSULTR Gene in Lathyrus sativus
Abstract:(1) To investigate the relationship between sulfate transporter genes (SULTR) and β N oxalyl L α,β diaminopropionicacid (β ODAP) content in Lathyrus sativus, we first identified 13 LsSULTR genes from transcriptomic database (SRP145030), which coding the four major groups of SULTR Ⅰ-Ⅳ separately. Therein, LsSULTR3; 3 and LsSULTR3; 5 were characterized by the conversed STAS (PF01740) and Sulfate_transp (PF00916) domain of SULTR, and gene expression levels of LsSULTR3; 3 and LsSULTR3; 5 strongly suggested relation with β ODAP biosynthesis. Bioinformatics analysis suggested that LsSULTR3; 3 and LsSULTR3; 5 can be regulated by various cis acting elements such as MYB and hormone response factors. In addition, protein protein interactions and protein phosphorylation also play an important role. (2) cDNAs of LsSULTR3; 3 and LsSULTR3; 5 were cloned with 1 962 bp encoding a peptide of 653 amino acid residues, and 1 923 bp encoding a peptide of 640 amino acid residues, respectively. (3) Semi quantity RT PCR analysis showed that LsSULTR3; 3 gene was expressed highest in stems and followed by main roots, old leaves, flowers, seeds in early flourishing podding stage (S2) and seedfilling flourishing stage (S6); while the LsSULTR3; 5 gene was expressed highest in flowers and followed by main roots, lateral roots and seeds in S2 stage. These results will help to reveal the mechanism of sulfur transport and assimilation, β ODAP biosynthesis in L. sativus.
keywords:Lathyrus sativus  sulfate transporter  β ODAP  gene cloning  protein activity regulation
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