大白菜橘紅心基因的精細定位及候選基因預測
發(fā)布時間：2018-08-19 15:26
【摘要】：橘紅心大白菜富含類胡蘿卜素,倍受消費者的青睞。大白菜橘紅心性狀是由一對隱性基因控制的質(zhì)量性狀。本研究選取橘紅心大白菜和普通白心大白菜為試材,對控制大白菜橘紅心性狀的目標基因進行精細定位,預測目標基因并分析目標基因的序列特征。利用熒光實時定量PCR技術(shù)對預測的橘紅心基因的表達模式和類胡蘿卜素生物合成過程中相關(guān)酶的表達量進行分析。在預測的目標基因啟動子區(qū)域開發(fā)分子標記,并在不同大白菜品種間驗證。主要研究結(jié)果如下：1.利用白心大白菜自交系Chiifu和橘紅心自交系07A163構(gòu)建了一個大規(guī)模F2分離群體,鑒別出2200株橘紅心個體作為精細定位群體,最終將橘紅心基因定位在A09染色體上SSR標記syau26和syau28之間,物理圖距約19.9kb。經(jīng)預測,該定位區(qū)間內(nèi)存在6個編碼基因,其中的Bra031539基因編碼類胡蘿卜素異構(gòu)酶,該酶是類胡蘿卜素生物合成過程中特定的異構(gòu)酶,因此將該基因作為控制大白菜橘紅心性狀的候選基因。2.白心大白菜品系Chiifu的Bra031539基因DNA全長3402 bp,含有13個外顯子,編碼含有589個氨基酸的蛋白。TargetP分析表明,Bra031539蛋白很可能為葉綠體運輸肽；Prosite蛋白質(zhì)序列模式分析表明,Bra031539蛋白含有多個修飾性位點：包括12個酪蛋白激酶2磷酸化位點、8個蛋白激酶磷酸化位點、1個cAMP和cGMP蛋白激酶磷酸化位點、13個N端豆蔻�；稽c、2個酪氨酸磷酸化位點、3個N端糖基化位點和1個亮氨酸拉鏈結(jié)構(gòu)。SMART分析表明,Bra031539蛋白存在4個結(jié)構(gòu)域：DAO結(jié)構(gòu)域、FAD_binding_2結(jié)構(gòu)域、NAD_binding_8結(jié)構(gòu)域和氨基氧化酶結(jié)構(gòu)域。3.測序分析表明,相對于白心Chiifu,橘紅心07A163材料的Bra031539基因在啟動子區(qū)域有88bp的堿基缺失、88個SNP位點,CDS1編碼區(qū)6個堿基的缺失導致2個氨基酸不能正常翻譯,43個SNP位點的突變導致15個氨基酸的改變,該基因在第13個編碼區(qū)開始發(fā)生移碼突變導致蛋白翻譯提前終止,并且在3’端非編碼區(qū)出現(xiàn)堿基的缺失。4.對三種不同球色大白菜品系的Bra031539基因進行克隆測序發(fā)現(xiàn),與白心材料和黃心材料相比,6份不同來源橘紅心材料的啟動子區(qū)域均發(fā)生88 bp和7 bp的堿基缺失,共有的SNP位點有30個；CDS內(nèi),6份橘紅心材料在CDS1有6個堿基的缺失,同時在編碼區(qū)共發(fā)生的SNP位點數(shù)有12個,3份橘紅心材料07A163、桔65和桔62同時發(fā)生的SNP位點數(shù)有15個,橘紅心材料中的07A163、桔65和桔62同時在CDS13末端發(fā)生移碼突變導致蛋白翻譯提前終止,并且3’端有堿基的缺失。5.根據(jù)不同球色材料在Bra031539啟動子區(qū)域設(shè)計的序列差異,開發(fā)出1個Indel標記-BrProl,該標記可以用于橘紅心性狀的分子標記輔助選擇。6.分析Bra031539基因在三種不同球色材料上的表達差異發(fā)現(xiàn),Bra031539基因在春化階段、苗期葉片、根部和莖部的相對表達量較低,且差異不顯著。受發(fā)育時期、表達器官和光信號調(diào)節(jié)的影響,該基因在大白菜結(jié)球期的葉片、花瓣上相對表達量較高且差異顯著。開花期是Bra031539基因相對表達量最高的時期,結(jié)球期的表達量隨著球葉從外向內(nèi)的順序逐漸降低,橘紅心07A163內(nèi)葉的相對表達量最低。7.通過對Chiifu和07A163的類胡蘿卜素生物合成相關(guān)基因的轉(zhuǎn)錄情況分析表明,春化階段,兩種材料之間的12個相關(guān)基因表達差異不顯著；受發(fā)育階段限制及表達器官影響,在苗期兩種材料間12個相關(guān)基因的表達差異也不明顯。然而,在苗期兩種材料的PSY基因則大量表達。8.類胡蘿卜素生物合成相關(guān)基因在兩種大白菜結(jié)球期的相對表達量差異顯著。在前期反應(yīng)中,催化各底物的酶IPI、GGPS、PSY、PDS和ZDS與白心材料相比表達量上調(diào),而后繼催化各種底物的酶如LCYB、LCYE、CHXB、ZEP、VDE、CCD和NCED與白心材料相比表達量下調(diào)。
[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.
【學位授予單位】：沈陽農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S634.1
，
本文編號：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.
【學位授予單位】：沈陽農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S634.1
，
本文編號：2192074
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