旨在建立稳定的银杏愈伤组织继代体系,筛选细胞活性强且黄酮产量高的细胞培养条件,为工业化生产提供一定参考.以MS培养基为基本培养基,在40 d的继代周期内设计正交实验研究外植体月份、外源激素与抗褐化剂交互作用下的银杏愈伤组织生长、褐化与黄酮积累,并对比了4、5、6月中旬银杏叶片黄酮含量.结果表明,银杏愈伤组织继代中的最佳生长组合为4月叶片诱导的愈伤组织+3.0 mg/L NAA+0.5 mg/L KT+5 mg/L VC;最佳抗褐化组合为4月叶片诱导的愈伤组织+1.0 mg/L NAA+1.0 mg/L KT+10 mg/L VC;银杏叶黄酮含量从4-6月呈上升趋势,且不同月份叶片诱导的愈伤组织黄酮含量与之呈正相关,以6月叶片诱导的愈伤组织+2.0 mg/L NAA+1.0 mg/L KT+5 mg/L VC为最佳黄酮积累条件;结合愈伤组织干重,总黄酮实际产量最高组合为5月叶片诱导的愈伤组织+2.0 mg/L NAA+0.5 mg/L KT+5 mg/L VC.银杏愈伤组织的适宜继代周期为24-32 d,生长量较大且褐化率低.

Objective The transcriptome sequencing was used to analyze the expression of key genes of flavonoid biosynthesis in different stages of growth and development of Ginkgo biloba. Methods The leaves of young trees and adult trees of G. biloba in different periods were used as the test material. Transcriptome sequencing analysis was carried out by using Illumina HiSeq 2000, and analyses of gene functional annotation of Unigene and expression characteristics of key genes for biosynthesis of G. biloba flavonoids were also performed. Results A total of 43 073 Unigene were obtained by transcriptome sequencing, of which 35 179 were annotated and 5 117 genes were screened by differential gene expression. Fifty candidate genes were screened by analyzing KEGG pathway related to flavonoid synthesis. The expression patterns of 50 candidate genes were analyzed. It was found that the key genes of flavonoid synthesis were all highly expressed in young leaves of G. biloba, but there was no significant difference in the leaves between adult and young trees at same time. The 13 genes closely related to the synthesis of flavonoids were analyzed. Among them, the expression of C4H, CHS, ANS, ANR, and FOMT genes was high, and the expression of F3'H, F3'5'H, and FLS genes was relatively low. Conclusion Through transcriptome sequencing, we screened and analyzed the key genes of flavonoid biosynthesis of G. biloba and their expression characteristics, which provided the theoretical basis of molecular pharmacology for improving the yield of ginkgo flavonoids.

The MADS-box gene is a family of genes that encode transcriptional regulators in eukaryotes and plays an important regulatory role in floral development. In order to study the function of MADS-box gene in G. biloba,We sequenced the transcriptome of G. biloba, including four stages of flower bud differentiation, and screened the MADS-box family of G. biloba. Expression pattern, sequence signature, subcellular location and phylogenetic analysis were analyzed by the bioinformatics methods. The results showed that, 15 G. biloba MADS-box family genes were obtained. According to the different types of expression patterns of differentially expressed genes, the target gene could be divided into 3 types. Analysis of the coding sequence of the target gene showed that the main components of 15 G. biloba MADS-box proteins were a-helix and nonregular coil. The subcellular localization was mainly in the nucleus. All the expressed products contained MADS domain. The newGb2734, Gb38883, Gb28587 and Gb33168 in the MADS-box gene family of G. biloba might play an important role in the flowering regulation of G. biloba. Gb16301 might be the key gene in the developmental process of G. biloba.

鉴定银杏花芽分化调控的关键基因,揭示银杏花芽分化调控的主要分子机制,为缩短银杏童期和选育银杏早花品种提供理论指导.本研究中采用高通量测序技术对银杏花芽分化3个时期(花芽未分化期、花芽分化始期、花芽分化盛期)的样品进行转录组测序,并分析数字表达谱,筛选开花调控相关基因并进行荧光定量PCR(RT-qPCR)表达验证.转录组测序共产生27.52 Gb原始数据,注释到8大功能数据库(GO、COG、KEGG、KOG、NR、Pfam、Swiss-Prot、eggNOG)上的unigene总数为35 179个.通过GO分类和KEGG Pathway富集性分析,将unigene分别归于55个GO类别和126个代谢途径.差异表达基因分析显示,花芽未分化期较花芽分化始期有2 253个基因上调,2 032个基因下调;花芽分化始期较花芽分化盛期有1 770个基因上调,1 901个基因下调;花芽未分化期较花芽分化盛期有1 865个基因上调,2 042个基因下调.发掘出大量的开花相关的基因涉及5个开花调控途径(光周期途径、春化途径、赤霉素途径、自主途径和年龄途径).筛选出gene.Gb_17 618(GI序列)、gene.Gb_19790 (FT/TFL1序列)、gene.Gb_16301(AG序列)、gene.Gb_28337(花发育MADS-box序列)、gene.Gb 01884 (SOC1序列)和gene.Gb_41704(CO序列)等6个银杏花芽分化差异表达关键基因序列,荧光定量PCR检测表达水平与转录组结果一致.

为阐明银杏开花调控机制,对不同时期的银杏叶片进行了转录组测序,通过分析开花前后表达量差异较大的基因,筛选出1个与开花调控相关的CONSTANS-LIKE基因.使用RT-PCR法从银杏叶片cDNA中克隆得到该基因的完整编码序列(CDS),命名为GbCOL.GbCOL基因的CDS序列全长为1 239 bp,编码产物含412个氨基酸.核苷酸和蛋白质序列多重比对结果显示,GbCOL与其他植物的CO基因的核苷酸与蛋白质序列具有一定的同源性,与裸子植物中的CO基因同源性较高.该基因的蛋白产物具有典型的CONSTANS蛋白保守结构,包含2个高度保守的B-box及CCT结构域.该基因可能在银杏开花调控过程中发挥重要作用.