芥菜型油菜种皮颜色的分子生物学研究
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摘要
与黑籽油菜相比，黄籽油菜以其种皮薄，纤维素、木质素含量低，蛋白质含量高，含油量高，油清澈透明及其饲用价值高等优点而受到油菜育种家的关注。芥菜型油菜种皮颜色遗传比较简单，黄籽对棕籽差别明显。为了弄清油菜黄籽的遗传规律和黄籽形成的分子基础，本研究以四川黄籽自交系和紫叶芥自交系及其杂种后代为材料，对芥菜型油菜种皮颜色和叶片颜色的遗传进行了研究；对控制棕色种皮的基因进行了RAPD标记；参照在拟南芥4-二氢黄酮还原酶基因序列，设计基因专一引物，靶向扩增克隆目的基因片段。
通过本研究，取得了以下的结果：
1 芥菜型油菜种皮颜色、叶片颜色的遗传分析表明，棕色种皮对黄色种皮为显性，BC_1和F_2代植株上种子种皮颜色出现分离，分离比分别为棕：黄为3：1和棕：黄为15：1，说明种皮颜色是由2对独立遗传基因控制的；叶片颜色是由一对基因控制的，紫色对绿色为显性。人工气候箱中的39株四川黄籽×(紫叶芥×四川黄籽)BC_1代叶色和种皮颜色分离情况研究表明：叶色基因和种皮颜色基因间的重组率为46.2％，初步判定芥菜型油菜种皮颜色基因和叶子颜色基因是独立遗传的。
2 培育1个位点显性、另一位点纯合隐性的棕籽近等基因系，已回交3代，即{[四川黄籽×(紫叶芥×四川黄籽)]×四川黄籽}×四川黄籽，获得了7个1个位点显性、另一位点隐性的棕籽材料，他们分别是Y04816，Y04818，Y04819，Y04820，Y04823，Y04825，Y04828。
3 以四川黄籽，紫叶芥，紫叶芥×四川黄籽F_1及四川黄籽×(紫叶芥×四川黄籽)BC_1单株为材料，利用群分法(bulked sergeant analysis，BSA)进行种皮颜色基因的定位。通过275个10-mer随机引物PCR分析，初步找到了与决定种皮颜色基因连锁的分子标记S20_(1000)，S73_(800)。
4 在芥菜型油菜四川黄籽自交系和紫叶芥自交系中克隆了DFR基因片段，并测序，二者该片段长度均为466碱基(base pair，bp)。并将这些序列在NCBI上用BLAST进行序列同源比较。四川黄籽DFR序列与甘蓝型油菜Stellar (Brassica napus)的二氢黄酮异构酶基因的同源性为99％(Expect=0.0)，可以认为该片段为四川黄籽二氢黄酮异构酶基因的片段；它与甘蓝(Brassica oleracea)、芜菁(Brssica rapa)的二氢黄酮异构酶基因的同源性分别为96％、93％；与白菜型油菜(Brassica carinata)、拟南芥(Arabidopsis thaliana)二氢黄酮还原酶基因的同源性分别为93％、90％。紫叶芥DFR序列与甘蓝型油菜Stellar的二氢黄酮异构酶基因的同源性为97％(Expect=0.0)，可以认为该片段为四川黄籽二氢黄酮异构酶基因的片段；它与甘蓝、芜菁的二氢黄酮异构酶基因的同源性分别为96％、94％；与白菜型油菜、拟南芥二氢黄酮还原酶基因的同源性分别为91％、90％。将四川黄籽DFR基因片段的序列提交Genebank登记，序列号为AY518296。
The breeding of yellow seed coat rapeseed are attracted increasing attention by rapeseed breeder. The yellow-seed trait is economically benefical to the oil industry, seeds of yellow cultivars have high oil content than those of dark-seed varieties. Yellow seeds of rapeseed have lower fiber and ligin, compared with those of dark-seed varieties. Yellow seed also have higher protein content than dark seeds. The seed coat color heredity in Brassicajuncea is simple, the yellow coat seed is obviously difference to the brown coat seed. In order to know the hereditary and molecular basis of yellow coat seed, we selected the materials which are Sichuan yellow selfing line and Ziyejie selfing line, we carried out the genetics studies of seed coat color and leaf color, RAPD(random amplified polymorphic DNA) analysis of gene which control the seed coat color. Using the primer designed from the gene of dihydroflavonol 4-reductase (DFR) in Arabidopsis thaliana, using the genomic DNA of Sichuan yellow Ziyejie as template, we obtained the DFR gene fragment. Through the research, we got some results as fellows:
1 We know the genetics of seed coat color and leaf color in Brassica juncea.the brown seed coat color was dominant character, the yellow seed coat color was recessive character, seed coat color was controlled by two genes, the functions of two genes were independent, the seed coat color in BC1 showed the segregation phenomenon, the ratio for segregation of brown seed and yellow seed is 3:1, the seed coat color in F2 showed the segregation phenomenon, the ratio for segregation of brown seed and yellow seed is 15:1. The leaf color was controlled by one gene, the brown leaf was dominant character, the green leaf was recessive character. The segregation phenomenon of seed coat color and leaf color of Sichuan yellow x (Ziyejie x Sichuan yellow) BC1 in growth house showed that the recombination frequency of seed coat color gene and leaf color gene was 46.2%, which suggest that the inheritance of seed coat color and leaf color was independent.
2 Breeding some lines that isolation of independent gene pairs at two loci for seed coat color in Brassicajuncea, one was dominant, the other was recessive. The lines are backcrossed to Sichuan yellow 3 times to obtain the lines that are {Sichuan yellow
X (Ziyejie X Sichuan yellow)} X Sichuan yellow.
3 Using Sichuan yellow, Ziyejie, Ziyejie X Sichuan yellow and Sichuan yellow X (Ziyejie X Sichuan yellow) as materials, we localized the seed coat color gene in Brassica jimcea. Using the way of bulked sergeant analysis(BSA), 275 10-mer random primers were chosen for polymorphism between the two bulks and Sichuan yellow, Ziyejie, we found two RAPD marker S201000, S73800 linked to brown seed coat color gene.
4 We cloned the DFR gene fragment in Sichuan yellow and Ziyejie, knowed
the sequences of the two DFR gene fragments, the length of DFR gene fragment in Sichuan yellow is same to this in Ziyejie, the length of the two DFR gene fragments are 466 base pair(bp). We compared the two sequences to NCBI-BLAST. In Sichuan yellow. 99% sequences are homologous to Brassica napus cultivar Stellar dihydroflavonone isomerase gene(partial cds), expect=0.0,we could consider the fragment was dihydroflavonone isomerase gene(partial cds) fragment; the dihydroflavonone isomerase gene( partial cds) sequences of Brassica oleracea cultivar and Brassica rapa cultivar have 96% and 93% homologization, the dihydroflavonol reductase gene (partial cds) sequences of Brassica carinata and Arabidopsis thaliana have 93% and 90% homologization, respectively, compared with Sichuan yellow DFR sequences. In Ziyejie, 97% sequences are homologous to Brassica napus cultivar Stellar dihydroflavonone isomerase gene(partial cds), expect=0.0,we could consider the fragment was dihydroflavonone isomerase gene(partial cds) fragment; the dihydroflavonone isomerase gene( partial cds) sequences of Brassica oleracea cultivar and Brassica rapa cultivar have 96% and 94% homologization. the dihydroflavonol reductase gene (partial cds) se
通过本研究，取得了以下的结果：
1 芥菜型油菜种皮颜色、叶片颜色的遗传分析表明，棕色种皮对黄色种皮为显性，BC_1和F_2代植株上种子种皮颜色出现分离，分离比分别为棕：黄为3：1和棕：黄为15：1，说明种皮颜色是由2对独立遗传基因控制的；叶片颜色是由一对基因控制的，紫色对绿色为显性。人工气候箱中的39株四川黄籽×(紫叶芥×四川黄籽)BC_1代叶色和种皮颜色分离情况研究表明：叶色基因和种皮颜色基因间的重组率为46.2％，初步判定芥菜型油菜种皮颜色基因和叶子颜色基因是独立遗传的。
2 培育1个位点显性、另一位点纯合隐性的棕籽近等基因系，已回交3代，即{[四川黄籽×(紫叶芥×四川黄籽)]×四川黄籽}×四川黄籽，获得了7个1个位点显性、另一位点隐性的棕籽材料，他们分别是Y04816，Y04818，Y04819，Y04820，Y04823，Y04825，Y04828。
3 以四川黄籽，紫叶芥，紫叶芥×四川黄籽F_1及四川黄籽×(紫叶芥×四川黄籽)BC_1单株为材料，利用群分法(bulked sergeant analysis，BSA)进行种皮颜色基因的定位。通过275个10-mer随机引物PCR分析，初步找到了与决定种皮颜色基因连锁的分子标记S20_(1000)，S73_(800)。
4 在芥菜型油菜四川黄籽自交系和紫叶芥自交系中克隆了DFR基因片段，并测序，二者该片段长度均为466碱基(base pair，bp)。并将这些序列在NCBI上用BLAST进行序列同源比较。四川黄籽DFR序列与甘蓝型油菜Stellar (Brassica napus)的二氢黄酮异构酶基因的同源性为99％(Expect=0.0)，可以认为该片段为四川黄籽二氢黄酮异构酶基因的片段；它与甘蓝(Brassica oleracea)、芜菁(Brssica rapa)的二氢黄酮异构酶基因的同源性分别为96％、93％；与白菜型油菜(Brassica carinata)、拟南芥(Arabidopsis thaliana)二氢黄酮还原酶基因的同源性分别为93％、90％。紫叶芥DFR序列与甘蓝型油菜Stellar的二氢黄酮异构酶基因的同源性为97％(Expect=0.0)，可以认为该片段为四川黄籽二氢黄酮异构酶基因的片段；它与甘蓝、芜菁的二氢黄酮异构酶基因的同源性分别为96％、94％；与白菜型油菜、拟南芥二氢黄酮还原酶基因的同源性分别为91％、90％。将四川黄籽DFR基因片段的序列提交Genebank登记，序列号为AY518296。
The breeding of yellow seed coat rapeseed are attracted increasing attention by rapeseed breeder. The yellow-seed trait is economically benefical to the oil industry, seeds of yellow cultivars have high oil content than those of dark-seed varieties. Yellow seeds of rapeseed have lower fiber and ligin, compared with those of dark-seed varieties. Yellow seed also have higher protein content than dark seeds. The seed coat color heredity in Brassicajuncea is simple, the yellow coat seed is obviously difference to the brown coat seed. In order to know the hereditary and molecular basis of yellow coat seed, we selected the materials which are Sichuan yellow selfing line and Ziyejie selfing line, we carried out the genetics studies of seed coat color and leaf color, RAPD(random amplified polymorphic DNA) analysis of gene which control the seed coat color. Using the primer designed from the gene of dihydroflavonol 4-reductase (DFR) in Arabidopsis thaliana, using the genomic DNA of Sichuan yellow Ziyejie as template, we obtained the DFR gene fragment. Through the research, we got some results as fellows:
1 We know the genetics of seed coat color and leaf color in Brassica juncea.the brown seed coat color was dominant character, the yellow seed coat color was recessive character, seed coat color was controlled by two genes, the functions of two genes were independent, the seed coat color in BC1 showed the segregation phenomenon, the ratio for segregation of brown seed and yellow seed is 3:1, the seed coat color in F2 showed the segregation phenomenon, the ratio for segregation of brown seed and yellow seed is 15:1. The leaf color was controlled by one gene, the brown leaf was dominant character, the green leaf was recessive character. The segregation phenomenon of seed coat color and leaf color of Sichuan yellow x (Ziyejie x Sichuan yellow) BC1 in growth house showed that the recombination frequency of seed coat color gene and leaf color gene was 46.2%, which suggest that the inheritance of seed coat color and leaf color was independent.
2 Breeding some lines that isolation of independent gene pairs at two loci for seed coat color in Brassicajuncea, one was dominant, the other was recessive. The lines are backcrossed to Sichuan yellow 3 times to obtain the lines that are {Sichuan yellow
X (Ziyejie X Sichuan yellow)} X Sichuan yellow.
3 Using Sichuan yellow, Ziyejie, Ziyejie X Sichuan yellow and Sichuan yellow X (Ziyejie X Sichuan yellow) as materials, we localized the seed coat color gene in Brassica jimcea. Using the way of bulked sergeant analysis(BSA), 275 10-mer random primers were chosen for polymorphism between the two bulks and Sichuan yellow, Ziyejie, we found two RAPD marker S201000, S73800 linked to brown seed coat color gene.
4 We cloned the DFR gene fragment in Sichuan yellow and Ziyejie, knowed
the sequences of the two DFR gene fragments, the length of DFR gene fragment in Sichuan yellow is same to this in Ziyejie, the length of the two DFR gene fragments are 466 base pair(bp). We compared the two sequences to NCBI-BLAST. In Sichuan yellow. 99% sequences are homologous to Brassica napus cultivar Stellar dihydroflavonone isomerase gene(partial cds), expect=0.0,we could consider the fragment was dihydroflavonone isomerase gene(partial cds) fragment; the dihydroflavonone isomerase gene( partial cds) sequences of Brassica oleracea cultivar and Brassica rapa cultivar have 96% and 93% homologization, the dihydroflavonol reductase gene (partial cds) sequences of Brassica carinata and Arabidopsis thaliana have 93% and 90% homologization, respectively, compared with Sichuan yellow DFR sequences. In Ziyejie, 97% sequences are homologous to Brassica napus cultivar Stellar dihydroflavonone isomerase gene(partial cds), expect=0.0,we could consider the fragment was dihydroflavonone isomerase gene(partial cds) fragment; the dihydroflavonone isomerase gene( partial cds) sequences of Brassica oleracea cultivar and Brassica rapa cultivar have 96% and 94% homologization. the dihydroflavonol reductase gene (partial cds) se
引文
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