蒺藜苜蓿豆球蛋白基因Mt1g072600启动子的克隆及器官表达分析  

Cloning of Promoter of Legumin Gene Mt1g072600 from Medicago truncatula and Organs Expression Analysis

作　　者：魏琦超[1] 王亚丽 周岩[1] WEI Qi-Chao;WANG Ya-Li;ZHOU Yan(School of Life Science and Technology,Henan Institute of Science and Technology,Xinxiang 453003,China)

机构地区：[1]河南科技学院生命科技学院,新乡453003

出　　处：《农业生物技术学报》2021年第10期1926-1935,共10页Journal of Agricultural Biotechnology

基　　金：河南省高等学校重点科研项目计划(17A180004);河南省重点研发与推广专项(202102110148)。

摘　　要：在改善种子的营养品质和利用种子生物反应器(seed-based bioreactor)生产有工业或药用价值的蛋白质等基因工程实践中,需要使用种子特异性启动子驱动外源基因的转录。种子贮藏蛋白(seed storage proteins, SSPs)为高等植物种子成熟过程中合成并贮存的大量贮藏蛋白质的总称,其基因的启动子是种子特异性启动子的重要来源。为发掘蒺藜苜蓿(Medicago truncatula)的种子特异性启动子,本研究克隆其主要贮藏蛋白豆球蛋白基因Mt1g072600编码区上游2 075 bp启动子序列(命名为:PMt1g072600),利用网站PLACE和PlantCARE预测顺式作用元件(cis-acting elements),发现其含有E-box、A/T rich element、P-box等多个种子特异性启动子相关顺式作用元件。构建其驱动β-葡萄糖苷酸酶(β-glucuronidase, GUS)基因的植物表达载体并进行拟南芥(Arabidopsis thaliana)的遗传转化,转基因拟南芥种子中GUS表达情况分析发现,Mt1g072600基因启动子PMt1g072600驱动GUS的表达量极显著地高于PCaMV35S,转基因拟南芥各组织/器官的组织化学染色结果表明PMt1g072600驱动GUS表现出种子表达的特异性。本研究克隆的蒺藜苜蓿来源的种子特异性启动子PMt1g072600,可用于驱动外源基因在转基因植物种子中特异性转录,为植物基因工程领域种子特异性启动子的选择和使用提供参考。In the practice of genetic engineering, such as improving the nutritional quality of seeds and using seed-based bioreactor to produce proteins with industrial or medicinal value, it is necessary to use seed specific promoters to drive the transcription of exogenous genes. Seed storage proteins are the general name of a large number of storage proteins synthesized and stored in the process of seed ripening in higher plants, the promoters of the genes coding seed storage proteins serve as an important source for seed specific promoters.In order to obtain the seed specific promoter of Medicago truncatula, 2 075 bp promoter sequence(named PMt1 g072600) of upstream of coding region of the major storage protein legumin gene Mt1 g072600 was cloned, and the putative cis-acting elements of the promoter were predicted using workflow embed in PLACE and PlantCARE, the results showed that this sequence contained E-box, A/T rich element, P-box and other cisacting elements with highly relevant features to seed specific promoter. The plant expression vector to drive the GUS gene using this promoter was constructed and transformed into Arabidopsis thaliana, the result of analysis of GUS expression level in transgenic A. thaliana seeds showed that the expression of GUS driven by PMt1 g072600 was significantly higher than that drived by PCaMV35 S. The results of histochemical staining of tissues/organs of transgenic A. thaliana showed that GUS driven by PMt1 g072600 showed the specificity of seed expression.This study identified a seed specific promoter PMt1 g072600 of M. truncatula, which can be used to drive the specific transcription of exogenous genes in transgenic plant seeds. This study also provides a reference for selection and utilization of seed specific promoters in plant genetic engineering.

关 键 词：蒺藜苜蓿 豆球蛋白 种子特异性启动子 

分 类 号：S541.9[农业科学—作物学]