玉米GRMZM2G455909基因的克隆及其抗病功能初步分析

doi:10.3969/j.issn.1004-1524.2022.09.16

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (9): 1976-984.DOI: 10.3969/j.issn.1004-1524.2022.09.16

玉米GRMZM2G455909基因的克隆及其抗病功能初步分析

徐莉^a(), 王其^b, 丁婷^b, 江腾^c^,^*()

a.安徽农业大学生命科学学院,安徽合肥 230061
b.安徽农业大学植物保护学院,安徽合肥 230061
c.安徽农业大学新农村发展研究院,安徽合肥 230061

收稿日期:2021-06-07 出版日期:2022-09-25 发布日期:2022-09-30
通讯作者: 江腾
作者简介:*江腾,E-mail: 15671565@qq.com
徐莉(1984—),女,安徽宁国人,硕士,从事生物学相关教学与试验室管理工作。E-mail: 61267156@qq.com
基金资助:
安徽省自然科学基金(2008085MC93)

Cloning of GRMZM2G455909 gene from maize and its functional analysis in transgenic plants

XU Li^a(), WANG Qi^b, DING Ting^b, JIANG Teng^c^,^*()

a. College of Life Sciences, Anhui Agricultural University,Hefei 230061, China
b. School of Plant Protection, Anhui Agricultural University,Hefei 230061, China
c. Institute of New Rural Development Research, Anhui Agricultural University,Hefei 230061, China

Received:2021-06-07 Online:2022-09-25 Published:2022-09-30
Contact: JIANG Teng

摘要/Abstract

摘要：

本课题组前期研究获得玉米GRMZM2G455909基因,该研究拟对GRMZM2G455909基因开展生物信息学分析,明确其分子特征;构建过表达载体pCAMBIA1301a-GRMZM2G455909,通过农杆菌介导法获得转GRMZM2G455909基因拟南芥植株,分析转GRMZM2G455909基因拟南芥植株抗病能力及其抗病信号通路。研究表明,GRMZM2G455909基因所编码的蛋白序列属于NBS-LRR类蛋白家族; Pst DC3000处理转GRMZM2G455909基因拟南芥植株,发现转基因植株体内菌体数量明显下降,其抗病性显著增强,推测Pst DC3000可能诱导转GRMZM2G45590基因拟南芥体内形成水杨酸抗病信号通路。研究结果可为作物遗传育种抗病性的改良提供新的路径。

关键词: GRMZM2G455909基因, 载体构建, 转基因拟南芥, 抗病性

Abstract:

The GRMZM2G455909 gene of maize was discovered in our previous study. In order to determine the molecular characteristics of this gene, bioinformatics analysis and a range of molecular experiments were carried out. The over-expression vector pCAMBIA1301A-GRMZM2G455909 was constructed and transformed into the Arabidopsis thaliana plants by an Agrobacterium-mediated method, then the disease-resistance and its signal pathway of transgenic Arabidopsis thaliana plants with GRMZM2G455909 gene were analyzed after inoculation with Pst DC3000. The results showed that the protein encoded by GRMZM2G455909 gene belonged to the NBS-LRR family, and the number of bacterium in leaves from transgenic Arabidopsis thaliana plants with GRMZM2G455909 gene was significantly decreased after inoculation with Pst DC3000 compared with wild-type plants. These suggested that transformation of Arabidopsis thaliana plants with GRMZM2G455909 gene might increase resistance to Pst DC3000 via a SA-mediated signaling pathway. This study could provide a new genetic and breeding approach for improving the disease resistance of crop.

Key words: GRMZM2G455909 gene, vector construction, transgenic Arabidopsis thaliana, disease-resistance

中图分类号:

S513

徐莉, 王其, 丁婷, 江腾. 玉米GRMZM2G455909基因的克隆及其抗病功能初步分析[J]. 浙江农业学报, 2022, 34(9): 1976-984.

XU Li, WANG Qi, DING Ting, JIANG Teng. Cloning of GRMZM2G455909 gene from maize and its functional analysis in transgenic plants[J]. Acta Agriculturae Zhejiangensis, 2022, 34(9): 1976-984.

图/表 10

表1 qRT-PCR引物序列

Table 1 Primers sequences used in qRT-PCR

基因 Gene	上游引物序列 Forward primer sequences(5'→3')	下游引物序列 Reverse primer sequences(5'→3')
AtActin2	GGTAACATTGTGCTCAGTGGTGG	AACGACCTTAATCTTCATGCT TGC
AtTUB4	CGAAAACGCTGACGAGTGTA	CCTTGGGAATGGGATAAGGT
GRMZM2G455909	CGACGAATGAGGCTCCTAGT	CTTGTCCAGCTCATTGTCGG

表2 抗病相关基因的引物序列

Table 2 The primers sequences of disease resistance related genes

基因 Gene	上游引物序列 Forward primer sequences(5'→3')	下游引物序列 Reverse primer sequences(5'→3')
PR1	ACACGTGCAATGGAGTTTGT	TGCAACTGATTATGGTTCCA
LOX	AGGAGTTTGGACGGGAGATT	CCGTACTTGCTCGGGTCA
EFR1	CCTTCCGAT CAA ATC CGT AAG	TCCCGAGCC AAA CCC TAA TAC
NPR1	AACGATTCTTCCCGCGCTGTTC	TTCTCCGCAAGCCAGTTGAGTC
AtActin2	GGTAACATTGTGCTCAGTGGTGG	AACGACCTTAATCTTCATGCT TGC
AtTUB4	CGAAAACGCTGACGAGTGTA	CCTTGGGAATGGGATAAGGT

图1 GRMZM2G455909同源蛋白氨基酸序列比对

Fig.1 Alignment of homologues amino acid sequences for amino acid sequence coded by GRMZM2G455909 gene

图2 GRMZM2G455909基因的结构(A)及其编码蛋白结构域(B)

Fig.2 GRMZM2G455909 gene structure(A) and its coding protein domain(B)

图3 GRMZM2G455909基因PCR扩增(A)及基因-Blunt Simple双酶切(B) M1为DL5000 DNA marker;1, 2为基因,CK为pEASY-GRMZM2G455909重组载体;3为双酶切结果。

Fig.3 Electrophoresis result of GRMZM2G455909 gene by PCR amplification(A) and gene-Blunt Simple by double enzyme digestion(B) M1 was DL5000 DNA marker; 1, 2 were both genes;CK was pEASY-Blunt Simple Cloning vector; 3 was the digestion result of transgenic plants.

图4 转基因阳性苗检测结果 M2为DL2000 DNA marker;L2,L9,转基因拟南芥植株。

Fig.4 Electrophoresis result of the detection for transgenic positive plants M2 was DL2000 DNA marker; L2,L9,Transgenic Arabidopsis plants.

图5 GRMZM2G455909转基因拟南芥阳性苗植株的qRT-PCR验证 WT, 野生型拟南芥植株;L2,L9,转基因拟南芥植株;不同的小写字母表示相对表达量在5%水平上差异显著。

Fig.5 Verification of transgenic Arabidopsis thaliana positive plants with GRMZM2G455909 gene by qRT-PCR WT, Wild Arabidopsis thaliana plant ; L2,L9,Transgenic Arabidopsis plants; Different lowercase letters indicated that the difference of relative expression at 5% level was obvious.

图6 接种Pst DC3000后转基因拟南芥发病情况 A,为第6 天时的表型;B,为不同时间的病情指数。WT,野生型拟南芥植株;L2,L9,转基因拟南芥植株。B图中同天不同处理不同的小写字母表示病指在5%水平上差异显著。

Fig.6 Incidence of transgenic Arabidopsis thaliana plants after inoculated with Pst DC3000 A was the phenotype at the 6 d; B was the disease index at different times. WT, Wild Arabidopsis thaliana plant ; L2,L9,Transgenic Arabidopsis plants.In B, different lowercase letters indicated that the difference of disease index at 5% level was obvious in different treatments on the same day.

表3 转基因拟南芥接种丁香假单胞菌DC3000后叶片体内菌体含量

Table 3 Number of bacterium in leaves from transgenic Arabidopsis thaliana plants after inoculation with Pst DC3000

处理 Treatments	时间 t/d	细菌数 Number of bacterium/(10⁵ CFU·g^-1)
WT	2	1.13 c
	4	11.23 a
	6	7.27 b
L2	2	0.57 c
	4	6.82 a
	6	1.58 b
L9	2	0.24 c
	4	3.22 a
	6	1.28 b

图7 转基因拟南芥中抗病相关基因的表达分析 WT,野生型拟南芥植株;L2和L9,转基因拟南芥植株。同一基因各处理间没有相同小写字母表示相对表达量在5%水平上差异显著。

Fig.7 Expression analysis of disease resistance related genes for transgenic Arabidopsis thaliana plants WT, Wild Arabidopsis thaliana plant ; L2 and L9,Transgenic Arabidopsis plants. Different lowercase letters indicated that the difference of relative expression at 5% level was obvious on the same gene from different treatments.

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玉米GRMZM2G455909基因的克隆及其抗病功能初步分析

Cloning of GRMZM2G455909 gene from maize and its functional analysis in transgenic plants

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