大白菜橘紅心基因的精細(xì)定位及候選基因預(yù)測(cè)
發(fā)布時(shí)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(fèi)者的青睞。大白菜橘紅心性狀是由一對(duì)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(duì)控制大白菜橘紅心性狀的目標(biāo)基因進(jìn)行精細(xì)定位,預(yù)測(cè)目標(biāo)基因并分析目標(biāo)基因的序列特征。利用熒光實(shí)時(shí)定量PCR技術(shù)對(duì)預(yù)測(cè)的橘紅心基因的表達(dá)模式和類胡蘿卜素生物合成過(guò)程中相關(guān)酶的表達(dá)量進(jìn)行分析。在預(yù)測(cè)的目標(biāo)基因啟動(dòng)子區(qū)域開(kāi)發(fā)分子標(biāo)記,并在不同大白菜品種間驗(yàn)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(gè)大規(guī)模F2分離群體,鑒別出2200株橘紅心個(gè)體作為精細(xì)定位群體,最終將橘紅心基因定位在A09染色體上SSR標(biāo)記syau26和syau28之間,物理圖距約19.9kb。經(jīng)預(yù)測(cè),該定位區(qū)間內(nèi)存在6個(gè)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(guò)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(zhǎng)3402 bp,含有13個(gè)外顯子,編碼含有589個(gè)氨基酸的蛋白。TargetP分析表明,Bra031539蛋白很可能為葉綠體運(yùn)輸肽；Prosite蛋白質(zhì)序列模式分析表明,Bra031539蛋白含有多個(gè)修飾性位點(diǎn)：包括12個(gè)酪蛋白激酶2磷酸化位點(diǎn)、8個(gè)蛋白激酶磷酸化位點(diǎn)、1個(gè)cAMP和cGMP蛋白激酶磷酸化位點(diǎn)、13個(gè)N端豆蔻�；稽c(diǎn)、2個(gè)酪氨酸磷酸化位點(diǎn)、3個(gè)N端糖基化位點(diǎn)和1個(gè)亮氨酸拉鏈結(jié)構(gòu)。SMART分析表明,Bra031539蛋白存在4個(gè)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(cè)序分析表明,相對(duì)于白心Chiifu,橘紅心07A163材料的Bra031539基因在啟動(dòng)子區(qū)域有88bp的堿基缺失、88個(gè)SNP位點(diǎn),CDS1編碼區(qū)6個(gè)堿基的缺失導(dǎo)致2個(gè)氨基酸不能正常翻譯,43個(gè)SNP位點(diǎn)的突變導(dǎo)致15個(gè)氨基酸的改變,該基因在第13個(gè)編碼區(qū)開(kāi)始發(fā)生移碼突變導(dǎo)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(duì)三種不同球色大白菜品系的Bra031539基因進(jìn)行克隆測(cè)序發(fā)現(xiàn),與白心材料和黃心材料相比,6份不同來(lái)源橘紅心材料的啟動(dòng)子區(qū)域均發(fā)生88 bp和7 bp的堿基缺失,共有的SNP位點(diǎn)有30個(gè)；CDS內(nèi),6份橘紅心材料在CDS1有6個(gè)堿基的缺失,同時(shí)在編碼區(qū)共發(fā)生的SNP位點(diǎn)數(shù)有12個(gè),3份橘紅心材料07A163、桔65和桔62同時(shí)發(fā)生的SNP位點(diǎn)數(shù)有15個(gè),橘紅心材料中的07A163、桔65和桔62同時(shí)在CDS13末端發(fā)生移碼突變導(dǎo)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(dòng)子區(qū)域設(shè)計(jì)的序列差異,開(kāi)發(fā)出1個(gè)Indel標(biāo)記-BrProl,該標(biāo)記可以用于橘紅心性狀的分子標(biāo)記輔助選擇。6.分析Bra031539基因在三種不同球色材料上的表達(dá)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(duì)表達(dá)量較低,且差異不顯著。受發(fā)育時(shí)期、表達(dá)器官和光信號(hào)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(duì)表達(dá)量較高且差異顯著。開(kāi)花期是Bra031539基因相對(duì)表達(dá)量最高的時(shí)期,結(jié)球期的表達(dá)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(duì)表達(dá)量最低。7.通過(guò)對(duì)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(gè)相關(guān)基因表達(dá)差異不顯著；受發(fā)育階段限制及表達(dá)器官影響,在苗期兩種材料間12個(gè)相關(guān)基因的表達(dá)差異也不明顯。然而,在苗期兩種材料的PSY基因則大量表達(dá)。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(duì)表達(dá)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(dá)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(dá)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(xué)位授予單位】：沈陽(yáng)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S634.1
，
本文編號(hào)：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(xué)位授予單位】：沈陽(yáng)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S634.1
，
本文編號(hào)：2192074
本文鏈接：http://sikaile.net/shoufeilunwen/nykjbs/2192074.html
最近更新
教材專著
