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

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