EMS诱导红阳猕猴桃耐寒突变体的筛选及转录组分析

Screening and transcriptome analysis of EMS-induced cold-tolerant mutants in Hongyang kiwifruit

关键词:  猕猴桃, 甲基磺酸乙酯(EMS), 耐寒突变体, 转录组, 热激蛋白

Key words:  kiwifruit, ethyl methanesulfonate(EMS), cold-tolerant mutant, transcriptome, heat shock protein

摘要:

红阳猕猴桃(Actinidia chinensis var. chinensis ‘Hongyang')具有较高的经济价值和营养价值,以及较好的市场开发前景。但近年红阳猕猴桃产区如云南、四川等多地多次遭遇倒春寒等极端天气,其抗寒性差的缺点限制了发展空间。该研究通过在组培的过程中使用甲基磺酸乙酯(EMS)诱导红阳猕猴桃突变体,进而筛选出耐寒突变体,并通过转录组分析探究其胁迫响应机制。该研究以红阳猕猴桃叶片为实验材料,在组培时(4.4 g·L^-1 MS+4.5 g·L^-1 琼脂+1.5 mg·L^-1 6-BA+0.1 mg·L^-1 NAA+15 g·L^-1 蔗糖+0.01～0.10 g·L^-1 EMS)利用EMS诱导技术诱导突变体,并在低温环境下筛选出耐寒突变体。选出的耐寒突变体和正常红阳猕猴桃组培苗先进行4 ℃ 12 h寒胁迫处理,再进行转录组测序分析。结果表明:(1)通过初步的表型鉴定,当EMS处理浓度为0.06 g·L^-1时诱导的部分突变体具有一定的耐寒性;(2)在转录组测序数据GO功能富集分析中,富集条目最多的是生物学过程;(3)利用KEGG数据库分析时,共筛选到21个差异表达基因在15条通路中得到注释且均为上调表达,其中内质网中的蛋白质加工通路(ath04141)中富集的差异表达基因最多,并且该通路内的sHSF、Hsp70和NEF可能与耐寒机制调控有关。综上研究结果为红阳猕猴桃耐寒种质资源的研究与利用提供了材料基础及理论依据。

Abstract:

The Hongyang kiwifruit (Actinidia chinensis var. chinensis ‘Hongyang')has high economic and nutritional values and good prospects for market development. However, in recent years Hongyang kiwifruit production areas such as Yunnan and Sichuan have been subjected to extreme weather such as inversions on several occasions, and its poor cold resistance has limited its scope for development. In this study, ethyl methanesulfonate(EMS)was used to induce mutants of Hongyang kiwifruit in a tissue culture process, which led to the screening of cold-tolerant mutants and the investigation of their stress response mechanisms through transcriptome analysis. In this study, mutants were induced using EMS induction technology using Hongyang kiwifruit leaves as experimental material in tissue culture(4.4 g·L^-1 MS + 4.5 g·L^-1 agar + 1.5 mg·L^-1 6-BA + 0.1 mg·L^-1 NAA + 15 g·L^-1 sucrose + 0.01-0.10 g·L^-1 EMS)and screened for cold-tolerant mutants under low temperature. Selected cold-tolerant mutants and normal Hongyang kiwifruit tissue culture seedlings were subjected to 4 ℃ 12 h cold stress treatment, while later, transcriptome sequencing analysis was performed. The results were as follows:(1)According to the preliminary phenotypic identification, some of the mutants induced by the 0.06 g·L^-1 EMS were phenotypically resistant to cold;(2)In the GO functional enrichment analysis of transcriptome sequencing data, the most enriched entries were in the biological processes;(3)When using KEGG database analysis, a total of 21 differentially expressed genes(DEGs)were annotated in 15 pathways, and which were all up-regulated. The protein processing pathway(ath04141)in the endoplasmic reticulum had the most DEGs, and sHSF, Hsp70, and NEF in this pathway may be related to the regulation of cold-tolerant mechanisms. The above findings provide a material basis and theoretical rationale for the research and utilization of cold-tolerant germplasm resources of Hongyang kiwifruit.