黑枸杞花青素合成代谢相关基因克隆及再生体系建立
发布时间：2018-10-31 18:48
【摘要】：黑枸杞(Lycium ruthenicum Murr)系茄科(Solanaceae)枸杞属(Lycium L.)植物,在传统医药中,常用于治疗心脏病、月经不调、更年期、高血压等疾病。黑枸杞果实富含的花青素,被认为是良好药理疗效的来源,但是其花青素积累的遗传机理以及组织再生的研究相对较少。本研究通过黑枸杞和红枸杞的果实高通量转录组测序和生物信息学分析,解析黑枸杞与红枸杞中与花青素代谢相关基因的表达或结构差异,并进行目标基因克隆及分析。同时外植体叶、茎建立野生黑枸杞的再生组织培养体系,为黑枸杞新品种选育提供良好的理论和实验基础。实验结果如下:1、RNA-seq在红枸杞和黑枸杞果实中组装了192,869个unigene,利用NR,Nt,Swissprot等蛋白数据库预测到152,209蛋白。58849个基因的表达量在红黑枸杞之间差异明显,其中33070个基因发生了上调,25779个基因的表达量发生了下调。72719个基因在KEGG代谢通路得到注释,“黄酮类生物合成”,“花青素生物合成”和“类胡萝卜素生物合成”代谢途径变化最大。花青素合成代谢相关基因PAL、C4H、C4L、CHS、CHI、F3H、F3'5'H、DFR、LDOX、MYB转录因子和bHLH转录因子在组装数据库中具有31个同源基因,但没有发现F3'H和3GT的同源基因。所有花青素合成代谢结构基因在黑枸杞中有较高的表达量,这说明其花青素的生物合成是比较活跃的。而所有MYB转录因子在黑枸杞中表达量显著高于红枸杞。MYB基因是调控花青素合成代谢代谢通路的调控因子,它们的转录可以导致花青素合成代谢结构基因的表达调控,很有可能是黑枸杞果实中积累花青素的原因。2、根据转录组测序的结果,分离到黑枸杞中的调控花青素合成的MYB基因SlAN2。该基因全长774bp,编码258个氨基酸,分子量为29775.84,等电点为7.79,包含33个负电荷残基数,34个正电荷残基数,属于SANT超基因家族。蛋白质疏水性最大值为1.322,最小值为-3.378,疏水平均值为-0.845,说明其具有一定的亲水性。跨膜区分析推测该蛋白质可能不具有跨膜区,主要是在膜外进行作用。分子进化分析表明SlAN2基因与辣椒、矮牵牛、茄子等物种中调控花青素合成代谢的MYB基因亲缘关系较近。3、在黑枸杞组织培养再生体系建立中,实验显示,MS+2,4-D 2.0 mg·L-1+KT1 mg·L-1为叶、茎外植体适宜的愈伤诱导培养基,出愈率分别达到96%和94%。分化培养时,MS+NAA 2.0 mg·L-1+6-BA 2.0 mg·L-1为适宜叶愈伤组织分化不定芽培养基,分化率达到94%,MS+NAA 1.5 mg·L-1+6-BA 2.0 mg·L-1为茎愈伤组织分化的适宜培养基,分化率达到89%。生根培养时,在MS+NAA 1 mg·L-1培养基中,黑枸杞不定芽的生根率达到最大值;叶愈伤组织和茎愈伤组织分化苗生根率分别为89%,86%。移栽实验中,叶外植体和茎外植体组织培养再生植株平均成活率分别为92%,88%。
[Abstract]:Black Lycium (Lycium ruthenicum Murr) line (Solanaceae) Lycium (Lycium L.) Plants, in traditional medicine, are often used to treat heart disease, irregular menstruation, menopause, hypertension and other diseases. The anthocyanin rich in black medlar fruit is considered to be the source of good pharmacological effect, but the genetic mechanism of anthocyanin accumulation and tissue regeneration are relatively few. In this study, high-throughput transcriptome sequencing and bioinformatics analysis were used to analyze the expression and structure differences of anthocyanin metabolism related genes between black medlar and red medlar, and to clone and analyze the target gene. At the same time, the regeneration tissue culture system of wild black Lycium barbarum L. was established by explant leaves and stems, which provided a good theoretical and experimental basis for the breeding of new black medlar varieties. The results were as follows: 1 unigene, was assembled in red medlar and black medlar fruit. Using NR,Nt,Swissprot and other protein databases, 152209 protein was predicted. The expression of 58849 genes was significantly different between black and red wolfberry. Of these, 33070 genes were up-regulated and 25779 genes were down-regulated. 72719 genes were annotated in the KEGG metabolic pathway, "flavonoid biosynthesis". The metabolic pathways of anthocyanin biosynthesis and carotenoid biosynthesis were the most varied. Anthocyanin biosynthesis related genes, PAL,C4H,C4L,CHS,CHI,F3H,F3'5'H,DFR,LDOX,MYB transcription factors and bHLH transcription factors, had 31 homologous genes in the assembly database, but no homologous genes of F3H and 3GT were found. All the anthocyanin biosynthesis structural genes were highly expressed in Lycium barbarum L., which indicated that the biosynthesis of anthocyanin was active. The expression of all MYB transcription factors in Lycium barbarum was significantly higher than that in Lycium barbarum L. MYB gene was the regulator of anthocyanin biosynthesis and metabolism pathway, and their transcription could lead to the regulation of anthocyanin biosynthesis structure gene expression. 2According to the results of transcriptome sequencing, the MYB gene SlAN2., which regulates anthocyanin synthesis, was isolated from Black Lycium barbarum L. The total length of the gene is 774 BP, which encodes 258 amino acids with molecular weight of 29775.84 and isoelectric point of 7.79, which contains 33 negative charge residues and 34 positive charge residues, belonging to the SANT supergene family. The maximum hydrophobicity of protein is 1.322, the minimum value is -3.378, and the average hydrophobic value is -0.845, which indicates that it has certain hydrophilicity. The transmembrane region analysis suggested that the protein might not have transmembrane region, but mainly acted outside the membrane. Molecular evolution analysis showed that SlAN2 gene was closely related to MYB gene which regulated anthocyanin biosynthesis in capsicum, petunia, eggplant and other species. 3. In the establishment of tissue culture and regeneration system of Black Lycium barbarum L., the experiment showed that, MS _ 2N _ 4-D 2.0 mg L ~ (-1) KT1 mg L ~ (-1) was a suitable callus induction medium for leaves and stem explants, with callus rates of 96% and 94%, respectively. , MS NAA 2.0 mg L-1 6-BA 2.0 mg L-1 was suitable for adventitious bud differentiation of leaf callus, and the differentiation rate was 94%. MS NAA 1.5 mg L-1 6-BA 2.0 mg L-1 was the best medium for callus differentiation, and the differentiation rate was 89%. The rooting rate of adventitious buds of Lycium barbarum L. was the highest in MS NAA 1 mg L-1 medium, and the rooting rate of leaf callus and stem callus differentiation seedling was 89%. In the transplanting experiment, the average survival rate of regenerated plants in tissue culture of leaf explants and stem explants was 92 and 8888, respectively.
【学位授予单位】：青海师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.19
[Abstract]:Black Lycium (Lycium ruthenicum Murr) line (Solanaceae) Lycium (Lycium L.) Plants, in traditional medicine, are often used to treat heart disease, irregular menstruation, menopause, hypertension and other diseases. The anthocyanin rich in black medlar fruit is considered to be the source of good pharmacological effect, but the genetic mechanism of anthocyanin accumulation and tissue regeneration are relatively few. In this study, high-throughput transcriptome sequencing and bioinformatics analysis were used to analyze the expression and structure differences of anthocyanin metabolism related genes between black medlar and red medlar, and to clone and analyze the target gene. At the same time, the regeneration tissue culture system of wild black Lycium barbarum L. was established by explant leaves and stems, which provided a good theoretical and experimental basis for the breeding of new black medlar varieties. The results were as follows: 1 unigene, was assembled in red medlar and black medlar fruit. Using NR,Nt,Swissprot and other protein databases, 152209 protein was predicted. The expression of 58849 genes was significantly different between black and red wolfberry. Of these, 33070 genes were up-regulated and 25779 genes were down-regulated. 72719 genes were annotated in the KEGG metabolic pathway, "flavonoid biosynthesis". The metabolic pathways of anthocyanin biosynthesis and carotenoid biosynthesis were the most varied. Anthocyanin biosynthesis related genes, PAL,C4H,C4L,CHS,CHI,F3H,F3'5'H,DFR,LDOX,MYB transcription factors and bHLH transcription factors, had 31 homologous genes in the assembly database, but no homologous genes of F3H and 3GT were found. All the anthocyanin biosynthesis structural genes were highly expressed in Lycium barbarum L., which indicated that the biosynthesis of anthocyanin was active. The expression of all MYB transcription factors in Lycium barbarum was significantly higher than that in Lycium barbarum L. MYB gene was the regulator of anthocyanin biosynthesis and metabolism pathway, and their transcription could lead to the regulation of anthocyanin biosynthesis structure gene expression. 2According to the results of transcriptome sequencing, the MYB gene SlAN2., which regulates anthocyanin synthesis, was isolated from Black Lycium barbarum L. The total length of the gene is 774 BP, which encodes 258 amino acids with molecular weight of 29775.84 and isoelectric point of 7.79, which contains 33 negative charge residues and 34 positive charge residues, belonging to the SANT supergene family. The maximum hydrophobicity of protein is 1.322, the minimum value is -3.378, and the average hydrophobic value is -0.845, which indicates that it has certain hydrophilicity. The transmembrane region analysis suggested that the protein might not have transmembrane region, but mainly acted outside the membrane. Molecular evolution analysis showed that SlAN2 gene was closely related to MYB gene which regulated anthocyanin biosynthesis in capsicum, petunia, eggplant and other species. 3. In the establishment of tissue culture and regeneration system of Black Lycium barbarum L., the experiment showed that, MS _ 2N _ 4-D 2.0 mg L ~ (-1) KT1 mg L ~ (-1) was a suitable callus induction medium for leaves and stem explants, with callus rates of 96% and 94%, respectively. , MS NAA 2.0 mg L-1 6-BA 2.0 mg L-1 was suitable for adventitious bud differentiation of leaf callus, and the differentiation rate was 94%. MS NAA 1.5 mg L-1 6-BA 2.0 mg L-1 was the best medium for callus differentiation, and the differentiation rate was 89%. The rooting rate of adventitious buds of Lycium barbarum L. was the highest in MS NAA 1 mg L-1 medium, and the rooting rate of leaf callus and stem callus differentiation seedling was 89%. In the transplanting experiment, the average survival rate of regenerated plants in tissue culture of leaf explants and stem explants was 92 and 8888, respectively.
【学位授予单位】：青海师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.19
【参考文献】
相关期刊论文 前10条
1 刘芳;唐映红;袁有美;郭清泉;沈帆;陈建荣;;多肉植物劳尔的组织培养[J];植物学报;2016年02期
2 杨宁;李宜s，
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