三明野生蕉抗寒机理解析及抗寒香蕉种质创新

Banana plants are generally low in cold resistance, and thus often suffer great yield losses in some of their cultivated regions of south China, such as in Guangdong, Guangxi and Fujian Provinces due to chilling or freezing injuries they might experience in winter and early spring. In our previous studies, the wild banana ‘Sanming’ was found to be very acclimate to cold, and it even can survive when temperature is lower than -5℃. In the proposed study, the cold resistant wild banana ‘Sanming’ and cold sensitive ‘Tianbaojiao’ will be firstly employed for cold related indices analysis before and after cold treatment to find out the physiological and biochemical mechanism of wild banana ‘Sanming’ cold resistance. Then, they will be subjected to RNA-Seq and whole genome bisulfite sequencing (WGBS) to compare their transcriptome and methylome change patterns under cold treatment and to construct a lncRNA-mRNA-miRNA-methylation regulation network. The obtained regulation network will contribute greatly to the cold resistance molecular mechanism clarification and cold-resistance related lncRNAs, mRNAs and miRNAs identification. Moreover, the putative cold resistance related transcripts will be used for over-expression and CRISPR/Cas9 targeted knockout vector construction and transformed into wild banana ‘Sanming’ and ‘Tianbaojiao’. The obtained transgenic banana plants will be subjected to cold resistance evaluation to demonstrate the transgenes’ function. The results obtained from this study will provide a solid basis for improving cold resistance of banana.

香蕉喜高温忌低温，然而我国广东、广西、福建等香蕉主产区时有冬春寒潮侵袭，严重影响了香蕉产业的健康发展。项目组在前期研究中发现三明野生蕉极为抗寒，甚至可耐受-5℃以下的低温。本项目拟以三明野生蕉和对寒害敏感的“天宝蕉”为研究材料，研究二者在冷处理前后的转录水平及全基因组甲基化水平上的差异。通过关联分析构建调控网络，结合抗寒相关生理生化指标，全面解析三明野生蕉抗寒机理，挖掘一些抗寒相关lncRNA、mRNA和miRNA。同时，构建抗寒相关转录本过量表达载体和CRISPR/Cas9基因敲除载体，通过遗传转化获得转基因三明野生蕉和“天宝蕉”并进行抗寒性评价。本研究结果有望为香蕉抗寒种质资源创新提供材料。

本项目以对寒害敏感的“天宝蕉”（TM）为对照，研究了抗寒香蕉种质三明野生蕉（SM）抗寒机制。形态学和生理生化研究结果显示SM抗寒特性与其较高的CTR、抗氧化酶活性和Pro含量有关。利用高通量测序技术比较了SM和TB在低温处理前后的转录本表达及全基因组甲基化水平上的差异。发现激素代谢、次生代谢等途径相关ncRNA和基因在SM抗寒反应中发挥重要作用，同时SM抗寒特性可能与其较低的本底甲基化水平有关。进一步通过比较SM在13℃、4℃、0℃，28℃下miRNAs组、lncRNA组差异情况，发现miR395、miR408、miR172等miRNAs、激素代谢和次生代谢相关基因受低温影响显著。克隆了SM部分miR172、miR408、miR395家族成员的前体以及香蕉IAA/AUX、CKII、ADA1、MPK1等基因，研究了它们在低温下的表达特性，发现它们都不同程度的参与香蕉低温胁迫响应。初步建立了香蕉基因枪法遗传转化体系，构建了4个重组载体，获得大量抗性芽。此外，本项目还通过叶绿体基因组分析明确了三明野生蕉遗传进化情况，同时对香蕉组培苗生根、移栽流程进行了一定的改进。在本项目的资助下，我们在BMC Plant Biology、Scientia Horticulturae、PLOS ONE、园艺学报、果树学报等杂志上发表论文8篇，培养了1名博士研究生和2名硕士研究生。