基于转录组测序的甘蔗野生种抗逆性相关基因克隆与分析

Sugarcane varieties were bred by interspecific hybridization in Saccharum. The main body of sugarcane genome comes from the original cultivar Saccharum officinarum L, and the genome also contains other wild species genetic materials, such as Saccharum spontaneum L. Because of the wild species genetic materials in sugarcane cultivar, sugarcane get stronger resistance to stresses than before, at the same time the planting areas expend from tropics to semi-tropics and temperate, and from paddy land to barrow and dry land. In order to increase sugarcane genome heterogeneity and broadening hereditary basis, we first successfully intergeneric distant hybridize Erianthus fulvus with sugarcane cultivar. Now we have got about 1000 new germplasm resources in different generations by intergeneric distant hybridization. In previous study, these germplasm resources show resistance to drought and cold, some characters liking wild specie Erianthus fulvus, but after generation by generation, with the wild special genetic materials cut down, the stress resistance ability become weak in some degree. In this research, we want to use RNA-Seq technology to find Erianthus fulvus special genes in these new germplasm resources, character their heredity structure and function, disclose their roles in gene expression and signal pathway, and explain heredity rule in different generations. Our results could make some efforts on parent and offspring selection and exogenous function genes separation, clone and transformation.

甘蔗栽培品种是由“甘蔗属”内的主要“种”通过“种间杂交”和“高贵化育种”选育而来。由于野生种抗逆性（主要是抗旱和耐寒）血缘的导入，使得甘蔗栽培品种的种植区域逐渐从热带向亚热带和温带、从水田向丘陵旱地扩展。所以，不断增强品种抗逆性是甘蔗育种的重要目标。为挖掘甘蔗野生种抗逆基因并探索其在栽培品种中所发挥的作用，本研究在前期工作基础上，拟采用RNA-Seq技术，以甘蔗主要野生种（割手密、蔗茅）及其后代栽培品种/系为供试材料，在其苗期经过干旱或低温胁迫后，进行转录组测序，筛选差异表达基因；然后通过功能注释、CGO、GO及KEGG分析，找到与抗旱或耐寒相关的候选基因；再进行RT-qPCR检测、克隆基因、构建表达载体、转化拟南芥、验证抗逆性等。研究结果既可探讨揭示甘蔗栽培品种中主要野生种血缘成分参与抗逆的作用与贡献，又可为进一步开展甘蔗抗逆基因的遗传转化提供理论依据。因而，具有重要科学意义和应用价值。

本研究采用转录组测序技术（RNA-Seq）挖掘甘蔗野生种及其后代栽培品种（系）中与抗旱、耐寒相关的功能基因，从转录表达的水平探讨揭示甘蔗栽培品种基因组中主要野生种血缘成分参与抗逆（抗旱、耐寒）的作用与贡献，并为开展甘蔗抗逆基因的遗传转化研究、提高甘蔗抗逆育种的效率和水平等提供理论依据和技术支撑，以解决甘蔗生产对抗逆新品种的迫切需要。以甘蔗近缘野生种（割手密、斑茅、蔗茅，等）和栽培种（热带种、印度种，等）及其杂交后代为供试材料，在其苗期经过干旱或低温胁迫后，进行转录组测序，筛选差异表达基因；然后通过功能注释、CGO、GO及KEGG分析，找到与抗旱或耐寒相关的候选基因；再进行RT-qPCR检测、克隆基因、构建表达载体、遗传转化、验证抗逆性等。通过实施，从“割手密”野生种中克隆到抗旱相关基因5个（ScNAC1、ScDREB、ScALDH、ScPP2C、SsWRKY1），从“斑茅”野生种中克隆到抗旱相关基因1个（SaWRKY），从“蔗茅”野生种中克隆到耐寒相关基因4个（ErCML30、ErDREB1A、EfNAC44、EfWRKY62）；完成了4个抗旱耐寒相关基因（ScNAC1、ScDREB、ErCML30、ErDREB1A）的遗传转化和功能验证；完成了基于叶绿体基因组测序的2个甘蔗原始亲本（热带种、割手密）的光合特性和耐寒性的比较分析；完成了甘蔗栽培品种与其原始亲本抗旱响应模式一致性的基因分析等。项目已发表论文13篇，其中SCI收录1篇；培养博士研究生2人、硕士研究生6名（均已毕业）；项目组成员中有1人获得硕士生导师资格。对照考核指标，已完成或超额完成了既定的研究目标。