甜瓜AMS基因结构分析及基因编辑载体构建

doi:10.6048/j.issn.1001-4330.2023.01.008

新疆农业科学 ›› 2023, Vol. 60 ›› Issue (1): 61-68.DOI: 10.6048/j.issn.1001-4330.2023.01.008

甜瓜AMS基因结构分析及基因编辑载体构建

才羿¹(), 戴冬洋¹^,², 谭海¹, 王迪³, 杨芬¹, 王岭¹, 盛云燕¹()

1.黑龙江八一农垦大学,黑龙江大庆 163319
2.石河子大学,新疆石河子 832000
3.黑龙江省农业科学院大庆分院,黑龙江大庆 163310

收稿日期:2022-05-09 出版日期:2023-01-20 发布日期:2023-03-07
通信作者: 盛云燕(1979-),女,黑龙江哈尔滨人,教授,硕士生导师,研究方向为瓜类分子遗传育种,(E-mail)shengyunyan@byau.edu.cn
作者简介:才羿(1997-),女,黑龙江齐齐哈尔人,硕士研究生,研究方向为瓜类分子遗传育种,(E-mail)1299234359@qq.com
基金资助:
国家自然科学基金“花药发育关键转录因子AMS调控甜瓜雄性不育机理研究”(31772330)

Analysis of Genetic Structure and Construction of Gene Editing Vector of Melon AMS Gene

CAI Yi¹(), DAI Dongyang¹^,², TAN Hai¹, WANG Di³, YANG Fen¹, WANG Ling¹, SHENG Yunyan¹()

1. Heilongjiang Bayi Agricultural University, Daqing Heilongjiang 163319, China
2. Shihezi University, Shihezi Xinjiang 832000, China
3. Daqing Branch of Heilongjiang Academy of Agricultural Science, Daqing Heilongjiang 163310, China

Received:2022-05-09 Published:2023-01-20 Online:2023-03-07
Correspondence author: SHENG Yunyan(1979-), female, Harbin City, Heilongjiang Province, Professor,Master Supervisor, research field: Molecular genetics and breeding of melons,(E-mail)shengyunyan@byau.edu.cn
Supported by:
National Science Foundation of China”Mechanism of AMS, a key transcription factor in anther development, regulating male sterility in Melon”(31772330)

摘要/Abstract

摘要：

【目的】研究鉴定甜瓜雄性不育候选基因ABORTED MICROSPORES(AMS)的基因家族,分析CRISPR基因编技术并构建其敲除载体,为甜瓜雄性不育基因AMS功能验证提供依据。【方法】采用生物信息学技术鉴定甜瓜雄性不育候选基因ABORTED MICROSPORES(AMS)家族基因,分析其进化关系、保守基序及理化性质。利用CRISPR/Cas9基因编辑技术以甜瓜AMS基因外显子区设计gRNA引物,以载体pHSN401为模板,扩增获得sgRNA克隆框,使用Bsa I酶切pHSN401载体,利用DNA重组酶构建重组载体并转化农杆菌,挑选单克隆进行培养,随后进行菌液PCR鉴定。【结果】甜瓜AMS基因编码的蛋白质长度从501~605 aa不等,平均分子量约为59 351 Da,等电点为5.04~6.45,甜瓜大部分AMS基因定位在细胞核;利用CRISPR/Cas9基因编辑技术,在AMS基因的CDS区设计3个靶位点,分别是AMS-pHSN401-1、AMS-pHSN401-2和AMS-pHSN401-3。【结论】AMS基因主要被预测在细胞核中表达,利用CRISPR/Cas9基因编辑技术构建甜瓜敲除重组载体AMS-pHSN401并转化农杆菌,获得阳性克隆。

关键词: 甜瓜; ABORTED MICROSPORES(AMS); 生物信息学; 载体构建

Abstract:

【Objective】 The construction of gene editing vector is an important step in gene function identification.Therefore, this article aims to study the gene family identification of ABORTED MICROSPORES (AMS), a candidate gene for male sterility in melon, explore CRISPR gene editing technology and construct its knockout vector, which is the function of melon gene, in the hope of providing a basis for AMS functional validation of the male infertility gene's melon. 【Methods】 In this study, bioinformatics technology was used to identify ABORTED MICROSPORES (AMS) family genes, which were candidate genes for male sterility in melon, and analyze their evolutionary relationships, conservative motifs, and physical and chemical properties.And CRISPR/Cas9 gene editing technology was applied to design gRNA primers in the exon region of the melon AMS gene, with the vector pHSN401 as a template to amplify the sgRNA cloning frame, with Bsa I to digest the pHSN401 vector, and DNA recombinase to construct the recombinant vector and transform it into agrobacterium.In addition to that, a single clone was selected for cultivation, and then PCR identification of bacterial liquid carried out. 【Results】 The results showed that the length of the protein encoded by the melon AMS gene ranged from 501-605 aa, the average molecular weight was about 59,351 Da, and the isoelectric point was between 5.04-6.45.Most of the melon AMS genes were located in the nucleus; CRISPR/Cas9 gene editing technology was used to design three target sites in the CDS region of the AMS gene, namely AMS-pHSN401-1, AMS-pHSN401-2 and AMS-pHSN401-3. 【Conclusion】 After analyzing the bioinformatics of muskmelon, the AMS gene is mainly predicted to be expressed in the nucleus, and the muskmelon knockout recombinant vector AMS-pHSN401 is successfully constructed by using CRISPR/Cas9 gene editing technology and transformed into agrobacterium, and the positive clone is obtained through the test.

Key words: melon; ABORTED MICROSPORES (AMS); bioinformatics; vector construction

中图分类号:

S652

才羿, 戴冬洋, 谭海, 王迪, 杨芬, 王岭, 盛云燕. 甜瓜AMS基因结构分析及基因编辑载体构建[J]. 新疆农业科学, 2023, 60(1): 61-68.

CAI Yi, DAI Dongyang, TAN Hai, WANG Di, YANG Fen, WANG Ling, SHENG Yunyan. Analysis of Genetic Structure and Construction of Gene Editing Vector of Melon AMS Gene[J]. Xinjiang Agricultural Sciences, 2023, 60(1): 61-68.

图/表 7

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引物名称 Primer name	引物序列 (5'-3') Primer sequence(5'-3')
sgAMS-1.F	ATTG CATCATGCAAATAAACCTTG
sgAMS-1.R	AAAC CAAGGTTTATTTGCATGATG
sgAMS-2.F	ATTG CCAACTTTTAGTACCCACAA
sgAMS-2.R	AAAC TTGTGGGTACTAAAAGTTGG
sgAMS-3.F	ATTG GGAGAGACTAAGACCCCTTG
sgAMS-3.R	AAAC CAAGGGGTCTTAGTCTCTCC
测序引物 Sequencing primer	GGATAAACCTTTTCACGCCC
鉴定引物F Identification Primer F	GGATAAACCTTTTCACGCCC
鉴定引物R Identification Primer R	ATTGAACGCCGAAGAACA

引物名称 Primer name	引物序列 (5'-3') Primer sequence(5'-3')
sgAMS-1.F	ATTG CATCATGCAAATAAACCTTG
sgAMS-1.R	AAAC CAAGGTTTATTTGCATGATG
sgAMS-2.F	ATTG CCAACTTTTAGTACCCACAA
sgAMS-2.R	AAAC TTGTGGGTACTAAAAGTTGG
sgAMS-3.F	ATTG GGAGAGACTAAGACCCCTTG
sgAMS-3.R	AAAC CAAGGGGTCTTAGTCTCTCC
测序引物 Sequencing primer	GGATAAACCTTTTCACGCCC
鉴定引物F Identification Primer F	GGATAAACCTTTTCACGCCC
鉴定引物R Identification Primer R	ATTGAACGCCGAAGAACA

基因ID Gene ID	氨基酸数目 Amino acid number/aa	分子量 Molecular weight /Da	等电点 Isoelectric point	亚细胞定位 Subcellular localization	染色体 Chromosome	染色体定位 Chromosomal location (bp)
MELO3C021653.2.1	605	68637.8	5.04	细胞核	Chr 09	5019154-5023770
MELO3C013851.2.1	537	58 489.77	6.45	细胞核	Chr 06	33991207-33993509
MELO3C024041.2.1	501	55 262.12	6.16	细胞核/胞间层	Chr 04	23240046-23242216
MELO3C003412.2.1	511	55 934.34	5.87	细胞核/细胞质	Chr 04	995627-998314
MELO3C006016.2.1	526	58 428.33	5.72	细胞核	Chr 06	590163-592617

基因ID Gene ID	氨基酸数目 Amino acid number/aa	分子量 Molecular weight /Da	等电点 Isoelectric point	亚细胞定位 Subcellular localization	染色体 Chromosome	染色体定位 Chromosomal location (bp)
MELO3C021653.2.1	605	68637.8	5.04	细胞核	Chr 09	5019154-5023770
MELO3C013851.2.1	537	58 489.77	6.45	细胞核	Chr 06	33991207-33993509
MELO3C024041.2.1	501	55 262.12	6.16	细胞核/胞间层	Chr 04	23240046-23242216
MELO3C003412.2.1	511	55 934.34	5.87	细胞核/细胞质	Chr 04	995627-998314
MELO3C006016.2.1	526	58 428.33	5.72	细胞核	Chr 06	590163-592617

基因ID Gene ID	α-螺旋 α-helix		延长链 Extended strand		β-折叠 β-sheet		无规则卷曲 Random coil
基因ID Gene ID	数量(个) Number	所占比例 Percentage(%)	数量(个) Number	所占比例 Percentage(%)	数量(个) Number	所占比例 Percentage(%)	数量(个) Number	所占比例 Percentage(%)
MELO3C021653.2.1	200	33.06	68	11.24	22	3.64	315	52.07
MELO3C013851.2.1	180	33.52	67	12.48	13	2.42	277	51.58
MELO3C024041.2.1	182	36.33	68	13.57	14	2.79	237	47.31
MELO3C003412.2.1	153	29.94	65	12.72	10	1.96	283	55.38
MELO3C006016.2.1	160	30.42	60	11.41	13	2.47	293	55.70

甜瓜AMS基因结构分析及基因编辑载体构建

Analysis of Genetic Structure and Construction of Gene Editing Vector of Melon AMS Gene

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