大白菜橘红心基因的精细定位及候选基因预测
发布时间：2018-08-19 15:26
【摘要】：橘红心大白菜富含类胡萝卜素,倍受消费者的青睐。大白菜橘红心性状是由一对隐性基因控制的质量性状。本研究选取橘红心大白菜和普通白心大白菜为试材,对控制大白菜橘红心性状的目标基因进行精细定位,预测目标基因并分析目标基因的序列特征。利用荧光实时定量PCR技术对预测的橘红心基因的表达模式和类胡萝卜素生物合成过程中相关酶的表达量进行分析。在预测的目标基因启动子区域开发分子标记,并在不同大白菜品种间验证。主要研究结果如下：1.利用白心大白菜自交系Chiifu和橘红心自交系07A163构建了一个大规模F2分离群体,鉴别出2200株橘红心个体作为精细定位群体,最终将橘红心基因定位在A09染色体上SSR标记syau26和syau28之间,物理图距约19.9kb。经预测,该定位区间内存在6个编码基因,其中的Bra031539基因编码类胡萝卜素异构酶,该酶是类胡萝卜素生物合成过程中特定的异构酶,因此将该基因作为控制大白菜橘红心性状的候选基因。2.白心大白菜品系Chiifu的Bra031539基因DNA全长3402 bp,含有13个外显子,编码含有589个氨基酸的蛋白。TargetP分析表明,Bra031539蛋白很可能为叶绿体运输肽；Prosite蛋白质序列模式分析表明,Bra031539蛋白含有多个修饰性位点：包括12个酪蛋白激酶2磷酸化位点、8个蛋白激酶磷酸化位点、1个cAMP和cGMP蛋白激酶磷酸化位点、13个N端豆蔻酰基化位点、2个酪氨酸磷酸化位点、3个N端糖基化位点和1个亮氨酸拉链结构。SMART分析表明,Bra031539蛋白存在4个结构域：DAO结构域、FAD_binding_2结构域、NAD_binding_8结构域和氨基氧化酶结构域。3.测序分析表明,相对于白心Chiifu,橘红心07A163材料的Bra031539基因在启动子区域有88bp的碱基缺失、88个SNP位点,CDS1编码区6个碱基的缺失导致2个氨基酸不能正常翻译,43个SNP位点的突变导致15个氨基酸的改变,该基因在第13个编码区开始发生移码突变导致蛋白翻译提前终止,并且在3’端非编码区出现碱基的缺失。4.对三种不同球色大白菜品系的Bra031539基因进行克隆测序发现,与白心材料和黄心材料相比,6份不同来源橘红心材料的启动子区域均发生88 bp和7 bp的碱基缺失,共有的SNP位点有30个；CDS内,6份橘红心材料在CDS1有6个碱基的缺失,同时在编码区共发生的SNP位点数有12个,3份橘红心材料07A163、桔65和桔62同时发生的SNP位点数有15个,橘红心材料中的07A163、桔65和桔62同时在CDS13末端发生移码突变导致蛋白翻译提前终止,并且3’端有碱基的缺失。5.根据不同球色材料在Bra031539启动子区域设计的序列差异,开发出1个Indel标记-BrProl,该标记可以用于橘红心性状的分子标记辅助选择。6.分析Bra031539基因在三种不同球色材料上的表达差异发现,Bra031539基因在春化阶段、苗期叶片、根部和茎部的相对表达量较低,且差异不显著。受发育时期、表达器官和光信号调节的影响,该基因在大白菜结球期的叶片、花瓣上相对表达量较高且差异显著。开花期是Bra031539基因相对表达量最高的时期,结球期的表达量随着球叶从外向内的顺序逐渐降低,橘红心07A163内叶的相对表达量最低。7.通过对Chiifu和07A163的类胡萝卜素生物合成相关基因的转录情况分析表明,春化阶段,两种材料之间的12个相关基因表达差异不显著；受发育阶段限制及表达器官影响,在苗期两种材料间12个相关基因的表达差异也不明显。然而,在苗期两种材料的PSY基因则大量表达。8.类胡萝卜素生物合成相关基因在两种大白菜结球期的相对表达量差异显著。在前期反应中,催化各底物的酶IPI、GGPS、PSY、PDS和ZDS与白心材料相比表达量上调,而后继催化各种底物的酶如LCYB、LCYE、CHXB、ZEP、VDE、CCD和NCED与白心材料相比表达量下调。
[Abstract]:Chinese cabbage with orange red heart is a kind of quality trait controlled by a pair of recessive genes. In this study, Chinese cabbage with orange red heart and Chinese cabbage with common cabbage were selected as test materials, and the target genes which controlled the character of Chinese cabbage with orange red heart were located, predicted and analyzed. Sequence characteristics of the target genes were analyzed by fluorescence real-time quantitative PCR. The predicted expression pattern of the orange heart gene and the expression of related enzymes during carotenoid biosynthesis were analyzed. Molecular markers were developed in the predicted target gene promoter region and validated among different Chinese cabbage varieties. The main results were as follows: 1. A large-scale F2 isolation population was constructed by using Chinese cabbage inbred line Chiifu and orange heart inbred line 07A163. 2200 orange heart individuals were identified as fine-mapping populations. Finally, the orange heart gene was mapped between SSR markers syau26 and syau28 on A09 chromosome, and the physical map distance was about 19.9 kb. The gene encoding Bra031539 encodes carotenoid isomerase, a specific isomerase in carotenoid biosynthesis, which is used as a candidate gene for controlling the red heart traits of Chinese cabbage. 2. The Bra031539 gene of Chinese cabbage strain Chiifu has a total length of 3402 BP and contains 13 exons. TargetP analysis showed that Bra031539 protein was probably a chloroplast transport peptide; Prosite protein sequence pattern analysis showed that Bra031539 protein contained multiple modification sites: 12 casein kinase 2 phosphorylation sites, 8 protein kinase phosphorylation sites, 1 cAMP and cGMP protein kinase phosphorylation site, 13. SMART analysis showed that Bra031539 protein had four domains: DAO domain, FAD_binding_2 domain, NAD_binding_8 domain and amino oxidase domain. Bra031539 gene of Red Heart 07A163 had 88 bp deletion in promoter region, 88 SNP loci, and 6 base deletions in CDS1 coding region, which resulted in the inability of two amino acids to translate properly. Mutations at 43 SNP loci resulted in the alteration of 15 amino acids. Bra031539 gene of three Chinese cabbage lines with different globular colors was cloned and sequenced. The results showed that 88 BP and 7 BP deletions occurred in the promoter region of six orange-red-heart materials from different sources, and 30 SNPs were found in the promoter region of six orange-red-heart materials, compared with white-heart materials and yellow-heart materials. There were 6 deletions in CDS1, 12 SNP loci in coding region, 3 in 07A163, 15 in 65and 62, and 107A163, 65and 62 in CDS13, resulting in the early termination of protein translation and the presence of base at 3'end. Base deletion. 5. An Indel marker, BrProl, was developed for marker-assisted selection of orange red heart traits based on the sequence differences of promoter regions in Bra031539 from different globular-colored materials. 6. Analysis of Bra031539 gene expression in three different globular-colored materials revealed that Bra031539 gene was expressed in vernalization and seedling leaves. The relative expression of Bra031539 gene in leaves and petals of Chinese cabbage during heading stage was higher and significantly different from that in petals under the influence of expression organs and light signal regulation. The relative expression of 12 genes related to carotenoid biosynthesis in Chiifu and 07A163 was the lowest. 7. The transcription analysis of the genes related to carotenoid biosynthesis in Chiifu and 07A163 showed that there was no significant difference in the expression of 12 genes related to carotenoid biosynthesis between the two materials at vernalization stage. There was no significant difference in the expression of 12 related genes between the two materials at the early stage. However, the PSY gene was overexpressed in the two materials at the seedling stage. 8. The relative expression of genes related to carotenoid biosynthesis was significantly different between the two Chinese cabbages at the heading stage. Specific expression was up-regulated, and then the expression of enzymes such as LCYB, LCYE, CHXB, ZEP, VDE, CCD and NCED, which catalyzed various substrates, was down-regulated.
【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S634.1
，
本文编号：2192074
[Abstract]:Chinese cabbage with orange red heart is a kind of quality trait controlled by a pair of recessive genes. In this study, Chinese cabbage with orange red heart and Chinese cabbage with common cabbage were selected as test materials, and the target genes which controlled the character of Chinese cabbage with orange red heart were located, predicted and analyzed. Sequence characteristics of the target genes were analyzed by fluorescence real-time quantitative PCR. The predicted expression pattern of the orange heart gene and the expression of related enzymes during carotenoid biosynthesis were analyzed. Molecular markers were developed in the predicted target gene promoter region and validated among different Chinese cabbage varieties. The main results were as follows: 1. A large-scale F2 isolation population was constructed by using Chinese cabbage inbred line Chiifu and orange heart inbred line 07A163. 2200 orange heart individuals were identified as fine-mapping populations. Finally, the orange heart gene was mapped between SSR markers syau26 and syau28 on A09 chromosome, and the physical map distance was about 19.9 kb. The gene encoding Bra031539 encodes carotenoid isomerase, a specific isomerase in carotenoid biosynthesis, which is used as a candidate gene for controlling the red heart traits of Chinese cabbage. 2. The Bra031539 gene of Chinese cabbage strain Chiifu has a total length of 3402 BP and contains 13 exons. TargetP analysis showed that Bra031539 protein was probably a chloroplast transport peptide; Prosite protein sequence pattern analysis showed that Bra031539 protein contained multiple modification sites: 12 casein kinase 2 phosphorylation sites, 8 protein kinase phosphorylation sites, 1 cAMP and cGMP protein kinase phosphorylation site, 13. SMART analysis showed that Bra031539 protein had four domains: DAO domain, FAD_binding_2 domain, NAD_binding_8 domain and amino oxidase domain. Bra031539 gene of Red Heart 07A163 had 88 bp deletion in promoter region, 88 SNP loci, and 6 base deletions in CDS1 coding region, which resulted in the inability of two amino acids to translate properly. Mutations at 43 SNP loci resulted in the alteration of 15 amino acids. Bra031539 gene of three Chinese cabbage lines with different globular colors was cloned and sequenced. The results showed that 88 BP and 7 BP deletions occurred in the promoter region of six orange-red-heart materials from different sources, and 30 SNPs were found in the promoter region of six orange-red-heart materials, compared with white-heart materials and yellow-heart materials. There were 6 deletions in CDS1, 12 SNP loci in coding region, 3 in 07A163, 15 in 65and 62, and 107A163, 65and 62 in CDS13, resulting in the early termination of protein translation and the presence of base at 3'end. Base deletion. 5. An Indel marker, BrProl, was developed for marker-assisted selection of orange red heart traits based on the sequence differences of promoter regions in Bra031539 from different globular-colored materials. 6. Analysis of Bra031539 gene expression in three different globular-colored materials revealed that Bra031539 gene was expressed in vernalization and seedling leaves. The relative expression of Bra031539 gene in leaves and petals of Chinese cabbage during heading stage was higher and significantly different from that in petals under the influence of expression organs and light signal regulation. The relative expression of 12 genes related to carotenoid biosynthesis in Chiifu and 07A163 was the lowest. 7. The transcription analysis of the genes related to carotenoid biosynthesis in Chiifu and 07A163 showed that there was no significant difference in the expression of 12 genes related to carotenoid biosynthesis between the two materials at vernalization stage. There was no significant difference in the expression of 12 related genes between the two materials at the early stage. However, the PSY gene was overexpressed in the two materials at the seedling stage. 8. The relative expression of genes related to carotenoid biosynthesis was significantly different between the two Chinese cabbages at the heading stage. Specific expression was up-regulated, and then the expression of enzymes such as LCYB, LCYE, CHXB, ZEP, VDE, CCD and NCED, which catalyzed various substrates, was down-regulated.
【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S634.1
，
本文编号：2192074
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