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作物学报 ›› 2021, Vol. 47 ›› Issue (6): 1197-1202.doi: 10.3724/SP.J.1006.2021.03037

• 研究简报 • 上一篇    

玉米光敏色素A1基因(ZmPHYA1)在棉花中的转化及分子鉴定

马燕斌1(), 王霞2, 李换丽1, 王平3, 张建诚1, 文晋1, 王新胜1, 宋梅芳3, 吴霞1,*(), 杨建平4,*()   

  1. 1山西农业大学(山西省农业科学院)棉花研究所, 山西运城 044000
    2运城学院生命科学系, 山西运城 044000
    3北京市辐射中心, 北京 100875
    4河南农业大学农学院, 河南郑州 450002
  • 收稿日期:2020-06-15 接受日期:2020-11-13 出版日期:2021-06-12 网络出版日期:2020-12-28
  • 通讯作者: 吴霞,杨建平
  • 作者简介:马燕斌, E-mail:myb0517@163.com
  • 基金资助:
    山西省重点研发计划子课题(201703D211007-3);国家自然科学基金项目(31201253);国家自然科学基金项目(31871709);国家转基因生物新品种培育重大专项(2016ZX08010-003);山西省农业科学院应用基础研究计划(YGJPY2007);运城学院博士科研启动项目(YQ-2017 008)

Transformation and molecular identification of maize phytochrome A1 gene (ZmPHYA1) in cotton

MA Yan-Bin1(), WANG Xia2, LI Huan-Li1, WANG Pin3, ZHANG Jian-Cheng1, WEN Jin1, WANG Xin-Sheng1, SONG Mei-Fang3, WU Xia1,*(), YANG Jian-Ping4,*()   

  1. 1Institute of Cotton Research, Shanxi Agricultural University (Shanxi Academy of Agricultural Sciences), Yuncheng 044000, Shanxi, China
    2Department of Life Science, Yuncheng University, Yuncheng 044000, Shanxi, China
    3Beijing Radiation Center, Beijing 100875, China
    4College of Agronomy, Henan Agriculture University, Zhengzhou 450002, Henan, China
  • Received:2020-06-15 Accepted:2020-11-13 Published:2021-06-12 Published online:2020-12-28
  • Contact: WU Xia,YANG Jian-Ping
  • Supported by:
    The Shanxi Province Key Research and Development Program(201703D211007-3);The National Natural Science Foundation of China(31201253);The National Natural Science Foundation of China(31871709);The National Major Project for Developing New GM Crops(2016ZX08010-003);The Basic Research Program of Agricultural Sciences of Shanxi Academy(YGJPY2007);The Doctoral Research Project of Yuncheng College(YQ-2017 008)

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摘要：

为评价玉米ZmPHYA1基因在棉花种质资源改良中的价值, 本研究利用农杆菌介导法在陆地棉(Gossypium hirsutum) R15材料中进行了玉米ZmPHYA1基因的遗传转化。经过愈伤组织诱导、抗性愈伤筛选、体细胞分化诱导后获得棉花转基因再生植株。通过田间草铵膦除草剂筛选鉴定抗性植株, 并利用PCR扩增其草铵膦抗性基因和目的基因ZmPHYA1进行分子鉴定, 发现阳性植株对草铵膦除草剂具较好抗性, 并可扩增到256 bp的草铵膦抗性基因和217 bp ZmPHYA1基因的特异条带。进一步通过免疫印迹检测表明, 3个不同转基因株系中外源ZmPHYA1基因可正常表达约170 kD大小的蛋白, 且在不同组织中该外源蛋白均可正常表达。此外, 对转基因植株的不同农艺性状分析表明, 转基因株系株高明显低于受体对照, 而铃重和纤维长度等性状无明显差异。本研究成功获得具有草铵膦抗性和外源ZmPHYA1基因的棉花新种质材料, 为进一步利用光敏色素基因创新种质资源提供了材料来源。

关键词: 陆地棉, 玉米光敏色素A1, 蛋白表达, 草铵膦抗性

Abstract:

In order to evaluate the potential value of maize phytochrome A1 gene (ZmPHYA1) in the improvement of cotton germplasm resources, we transferred it into upland cotton (Gossypium hirsutum L.) R15 via Agrobacterium tumefaciens-mediated transformation with glufosinate-resistance gene as selection marker. The regenerated cotton plants were obtained through callus induction, antibiotic resistance screening and differentiation induction. After screening the regenerated plants by the herbicide glufosinate ammonium in the field, PCR detection confirmed that both the target bands, including 256 bp of the glufosinate gene and 217 bp band of ZmPHYA1 gene, were detected in the homozygous transgenic plants. In addition, the exogenous ZmPHYA1 protein of about 170 kD was also checked by immuno-blot in three transgenic cotton lines. The results showed that the specific proteins could be detected in different tissues, including leaves, flowers and stems in the transgenic Line 9. The plant height of transgenic Line 9, Line 14, Line 41 were significantly shorter than that of the wild type, while the differences of other yield-related agronomic traits were not observed between the transgenic lines and the wild type. In this study, new cotton germplasms with glufosinate resistance and ZmPHYA1 gene were successfully obtained, which provided a material source for further utilization of phytochrome gene to innovate germplasm resources.

Key words: Gossypium hirsutum, maize phytochrome A1, protein expression, glufosinate resistance

表1

转基因棉花植株中用于目标基因的检测引物"

引物
Primers		 序列
Primer sequences (5′-3′)		 片段长度
Expected size (bp)
Basta-F ACCATCGTCAACCACTACATC 256
Basta-R GCTGCCAGAAA CCCACGTCAT
ZmPHYA1-1641F CAGCAGAAGGATGCACCCTAGGCTG 217
ZmPHYA1-1857R CGCTTGCAGTTCGGCGAGCCCATCA

图1

外源ZmPHYA1 基因的棉花转化与再生植株的草铵膦抗性基因检测 A: 农杆菌侵染后的愈伤筛选及诱导; B: 愈伤诱导增殖; C: 胚型愈伤分化形成的体细胞胚胎; D: 再生的幼苗; E: 再生的嫁接植株; F: 部分再生株草铵膦抗性基因的检测, 其中1、2、4分别为株系Line 9、Line 14和Line 41, 3为非转基因的再生株, “-”为受体对照, “+”为质粒阳性扩增, M: DNA marker。"

图2

转外源基因ZmPHYA1棉花草铵膦抗性株系的鉴定与PCR分子检测 A, B: 抗除草剂检测的不同阳性植株后代; C: 分离的非抗性植株后代; D: 部分抗性植株的ZmPHYA1 基因的PCR检测, 其中“+”为质粒阳性扩增, 1和2为Line 9株系的不同后代单株, 3和4为Line 14株系的不同后代单株, 5和6为Line 41株系的不同后代单株。“-”为受体对照; M: DNA marker。"

图3

不同棉花转基因株系中外源ZmPHYA1蛋白表达的免疫印迹检测 M: 蛋白marker; WT: 转基因棉花受体R15; Isolated non-transgenic line: 转基因株系分离的非抗性株; #9、#14、#41分别为转外源ZmphyA1基因的不同株系Line 9、Line 14和Line 41。“+”为检测中利用转玉米中同源基因ZmPHYA2 (3393 bp)的阳性棉花植株。"

图4

转基因植株不同农艺性状的分析比较 A: 转基因株系的株高表型, 标尺为10 cm; B依次为WT、#9 (Line 9), #14 (Line 14), #41 (Line 41)株系纤维长度图, 标尺为1 cm; C、D、E和F分别为WT、#9 (Line 9)、#14 (Line 14)、#41 (Line 41)的株系平均株高、平均单铃籽棉重、衣分以及纤维长度。*表示P < 0.05差异显著。"

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