黑枸杞花青素合成代謝相關(guān)基因克隆及再生體系建立
發(fā)布時間：2018-10-31 18:48
【摘要】：黑枸杞(Lycium ruthenicum Murr)系茄科(Solanaceae)枸杞屬(Lycium L.)植物,在傳統(tǒng)醫(yī)藥中,常用于治療心臟病、月經(jīng)不調(diào)、更年期、高血壓等疾病。黑枸杞果實富含的花青素,被認(rèn)為是良好藥理療效的來源,但是其花青素積累的遺傳機理以及組織再生的研究相對較少。本研究通過黑枸杞和紅枸杞的果實高通量轉(zhuǎn)錄組測序和生物信息學(xué)分析,解析黑枸杞與紅枸杞中與花青素代謝相關(guān)基因的表達(dá)或結(jié)構(gòu)差異,并進(jìn)行目標(biāo)基因克隆及分析。同時外植體葉、莖建立野生黑枸杞的再生組織培養(yǎng)體系,為黑枸杞新品種選育提供良好的理論和實驗基礎(chǔ)。實驗結(jié)果如下:1、RNA-seq在紅枸杞和黑枸杞果實中組裝了192,869個unigene,利用NR,Nt,Swissprot等蛋白數(shù)據(jù)庫預(yù)測到152,209蛋白。58849個基因的表達(dá)量在紅黑枸杞之間差異明顯,其中33070個基因發(fā)生了上調(diào),25779個基因的表達(dá)量發(fā)生了下調(diào)。72719個基因在KEGG代謝通路得到注釋,“黃酮類生物合成”,“花青素生物合成”和“類胡蘿卜素生物合成”代謝途徑變化最大�；ㄇ嗨睾铣纱x相關(guān)基因PAL、C4H、C4L、CHS、CHI、F3H、F3'5'H、DFR、LDOX、MYB轉(zhuǎn)錄因子和bHLH轉(zhuǎn)錄因子在組裝數(shù)據(jù)庫中具有31個同源基因,但沒有發(fā)現(xiàn)F3'H和3GT的同源基因。所有花青素合成代謝結(jié)構(gòu)基因在黑枸杞中有較高的表達(dá)量,這說明其花青素的生物合成是比較活躍的。而所有MYB轉(zhuǎn)錄因子在黑枸杞中表達(dá)量顯著高于紅枸杞。MYB基因是調(diào)控花青素合成代謝代謝通路的調(diào)控因子,它們的轉(zhuǎn)錄可以導(dǎo)致花青素合成代謝結(jié)構(gòu)基因的表達(dá)調(diào)控,很有可能是黑枸杞果實中積累花青素的原因。2、根據(jù)轉(zhuǎn)錄組測序的結(jié)果,分離到黑枸杞中的調(diào)控花青素合成的MYB基因SlAN2。該基因全長774bp,編碼258個氨基酸,分子量為29775.84,等電點為7.79,包含33個負(fù)電荷殘基數(shù),34個正電荷殘基數(shù),屬于SANT超基因家族。蛋白質(zhì)疏水性最大值為1.322,最小值為-3.378,疏水平均值為-0.845,說明其具有一定的親水性�？缒^(qū)分析推測該蛋白質(zhì)可能不具有跨膜區(qū),主要是在膜外進(jìn)行作用。分子進(jìn)化分析表明SlAN2基因與辣椒、矮牽牛、茄子等物種中調(diào)控花青素合成代謝的MYB基因親緣關(guān)系較近。3、在黑枸杞組織培養(yǎng)再生體系建立中,實驗顯示,MS+2,4-D 2.0 mg·L-1+KT1 mg·L-1為葉、莖外植體適宜的愈傷誘導(dǎo)培養(yǎng)基,出愈率分別達(dá)到96%和94%。分化培養(yǎng)時,MS+NAA 2.0 mg·L-1+6-BA 2.0 mg·L-1為適宜葉愈傷組織分化不定芽培養(yǎng)基,分化率達(dá)到94%,MS+NAA 1.5 mg·L-1+6-BA 2.0 mg·L-1為莖愈傷組織分化的適宜培養(yǎng)基,分化率達(dá)到89%。生根培養(yǎng)時,在MS+NAA 1 mg·L-1培養(yǎng)基中,黑枸杞不定芽的生根率達(dá)到最大值;葉愈傷組織和莖愈傷組織分化苗生根率分別為89%,86%。移栽實驗中,葉外植體和莖外植體組織培養(yǎng)再生植株平均成活率分別為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.
【學(xué)位授予單位】：青海師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：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.
【學(xué)位授予單位】：青海師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S567.19
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 劉芳;唐映紅;袁有美;郭清泉;沈帆;陳建榮;;多肉植物勞爾的組織培養(yǎng)[J];植物學(xué)報;2016年02期
2 楊寧;李宜s，
本文編號：2303210
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