Mechlppdk基因在木薯中的表达分析及RNA干扰载体构建和遗传转化

doi:10.11924/j.issn.1000-6850.casb2020-0359

中国农学通报 ›› 2021, Vol. 37 ›› Issue (17): 19-25.doi: 10.11924/j.issn.1000-6850.casb2020-0359

所属专题：生物技术；马铃薯

Mechlppdk基因在木薯中的表达分析及RNA干扰载体构建和遗传转化

王海燕¹(), 陈新¹, 周新成¹, 沈旭², 孔华¹, 王文泉³()

¹中国热带农业科学院热带生物技术研究所,海口 571101
²南京农业大学生命科学院,南京 210095
³海南大学热带作物学院,海口 570228

收稿日期:2020-08-14 修回日期:2020-11-16 出版日期:2021-06-15 发布日期:2021-06-29
通讯作者: 王文泉
作者简介:王海燕,女,1977年出生,山东泰安人,助理研究员,博士,研究方向：热带作物基因工程。通信地址：271101 海南省海口市学院路4号中国热带农业科学院热带生物技术研究所,Tel：0898-66894533,E-mail：wanghaiyan@itbb.org.cn。
基金资助:
国家自然科学基金“木薯UGT85K4基因在培育抗旱和低氰木薯中功能的研究”(31701509);国家自然科学基金“基于CRISPR/Cas9 技术获得高直、支链淀粉木薯新种质的研究”(31771883)

Expression Analysis of Mechlppdk Gene in Cassava and Construction of RNA Interference Vector and Genetic Transformation

Wang Haiyan¹(), Chen Xin¹, Zhou Xincheng¹, Shen Xu², Kong Hua¹, Wang Wenquan³()

¹The Institute of Tropical Bioscience and Biotechnology, CATAS, Haikou, Hainan 571101
²College of Life Science, Nanjing Agricultural University, Nanjing 210095
³College of Tropical Crops, Hainan University, Haikou 570228

Received:2020-08-14 Revised:2020-11-16 Online:2021-06-15 Published:2021-06-29
Contact: Wang Wenquan

摘要/Abstract

摘要：

本研究为了明确丙酮酸磷酸双激酶(ppdk)在木薯中的表达谱,创制Mechlppdk的干扰株系,以期为Mechlppdk的功能研究提供材料。本研究以栽培型木薯Arg7为材料,用qPCR的方法分析Mechlppdk在木薯根、茎、叶中的表达谱;采用RNA干扰的方法,构建Mechlppdk的干扰载体,并遗传转化木薯脆性愈伤,经木薯再生体系获得转基因苗,在添加抗生素的培养基上进行生根筛选,经PCR鉴定获得Mechlppdk的干扰株系。用qPCR的方法鉴定Mechlppdk在干扰株系的表达情况。结果表明,Mechlppdk在木薯叶片中表达量最高,达到管家基因的50%。在Mechlppdk的转运肽区,选取1个保守区段设计引物,构建RNA干扰表达载体pART27-Mechlppdki,将表达载体转化农杆菌LBA4404,侵染木薯脆性胚愈伤,获得阳性转基因木薯苗7个株系。通过qPCR分析发现各株系Mechlppdk转录本有不同程度的降低。通过以上分析表明本研究获得了干扰效率较高木薯Mechlppdk干扰株系,将为解析木薯Mechlppdk的功能提供研究材料。

关键词: 丙酮酸磷酸双激酶, 表达谱, RNA干扰, 表达载体, 木薯, 遗传转化, 脆性愈伤, 转基因苗, PCR鉴定

Abstract:

The aim of this study is to clarify the expression profiles of pyruvate phosphodikinase (ppdk) in cassava and to create an interfering strain of Mechlppdk, so as to provide materials for the functional study of Mechlppdk. The cultivated cassava Arg7 was used as the research material. The expression profiles of Mechlppdk in cassava root, stem and leaf were analyzed by qPCR method; the interference vector of Mechlppdk was constructed by RNA interference method, and then transformed into friable callus of cassava. The transgenic plantlets were obtained by cassava regeneration system, and rooting screening was carried out on the medium supplemented with antibiotics. The interference lines of Mechlppdk were identified by PCR. The expression of Mechlppdk in the interference strains were identified by qPCR. The results showed that the highest expression level of Mechlppdk was found in cassava leaves, reaching 50% of housekeeper gene. In the transit peptide region of Mechlppdk, a conservative region was selected to design primers to construct the RNA interference expression vector pART27-Mechlppdki. The expression vector was transformed into Agrobacterium tumefaciens LBA4404 and used to infect the callus of cassava friable callus, and seven positive transgenic cassava lines were obtained. qPCR showed that the transcripts of Mechlppdk decreased in different degrees. In this study, we have obtained the cassava Mechlppdk interference lines with higher interference efficiency, which could provide research materials for analyzing the function of Mechlppdk.

Key words: pyruvate phosphate dikinase, expression profile, RNA interference, expression vector, Manihot esculenta, genetic transformation, friable callus, transgenic plantlets, PCR identification

中图分类号:

S533

王海燕, 陈新, 周新成, 沈旭, 孔华, 王文泉. Mechlppdk基因在木薯中的表达分析及RNA干扰载体构建和遗传转化[J]. 中国农学通报, 2021, 37(17): 19-25.

Wang Haiyan, Chen Xin, Zhou Xincheng, Shen Xu, Kong Hua, Wang Wenquan. Expression Analysis of Mechlppdk Gene in Cassava and Construction of RNA Interference Vector and Genetic Transformation[J]. Chinese Agricultural Science Bulletin, 2021, 37(17): 19-25.

图/表 8

图1. Mechlppdk RNAi干扰载体构建流程图红色代表正义链,绿色代表反义链。蓝色箭头指两个反向互补片段在载体中的插入位置

图2. Mechlppdk在根、茎、叶中的相对表达量

图3. 木薯Mechlppdk siRNA设计 A：特异片段正义序列和反义序列;B：正义和反义序列中间添加intron折叠的二级结构;下划线为引物序列。橙色字母为添加的酶切位点序列

图4. Mechlppdk RNA干扰片段的扩增及克隆鉴定 A：Mechlppdki特异片段的PCR扩增;1：MechlppdkiXE的PCR扩增,2：MechlppdkiCX的PCR扩增; B：阳性克隆的PCR鉴定;M2000：D2000 DNA ladder

图5. pHANNIBAL-MechlppdkiXE-CX中间载体酶切鉴定 A pHANNIBAL-MechlppdkiXE经Xho I/EcoR I酶切鉴定;B pHANNIBAL-MechlppdkiXE-CX经Cla I/Xba I酶切鉴定;M2000：D2000 DNA ladder

图6. pART27-MechlppdkRNAi的分子鉴定 A：pART27-MechlppdkRNAi PCR鉴定;B：pART27-MechlppdkRNAi NotI 酶切鉴定;M2000：D2000 DNA ladder;M15000：D15000 DNA ladder

图7. 转pART27-MechlppdkRNAi木薯苗的诱导及鉴定 A：农杆菌侵染木薯脆性胚愈伤;B：子叶的诱导;C：植株诱导;D：卡那霉素(50 μg/L)的生根筛选;E转基因木薯的筛选基因Npt II的PCR检测

图8. Mechlppdk干扰株系Mechlppdk的相对表达分析此处的表达量是指与野生型木薯中Mechlppdk的表达量相对比的比值

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Mechlppdk基因在木薯中的表达分析及RNA干扰载体构建和遗传转化

Expression Analysis of Mechlppdk Gene in Cassava and Construction of RNA Interference Vector and Genetic Transformation

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