木本油料植物星油藤形态发生基因BABY BOOM的克隆及其功能研究

Cloning and Functional Analysis of Morphogenetic Gene BABY BOOM in the Woody Oil Plant Plukenetia volubilis L.

星油藤(Plukenetia volubilis L.)是一种极具开发前景的木本油料作物,但尚未建立完整有效的遗传转化体系,这严重地制约了星油藤分子育种技术的应用进程。BABY BOOM(BBM)是广泛存在于植物中的转录因子,被认为在植物胚胎发育与植物再生中起到重要的调控作用。本研究从星油藤中克隆了拟南芥(Arabidopsis thaliana)AtBBM的同源基因PvoBBM,并对其表达特性和功能进行了研究,为利用该基因促进星油藤转基因育种奠定基础。利用本地BLAST从星油藤基因组序列中筛选AINTEGUMENTA-LIKE(AIL)基因家族成员,通过序列比对和系统进化鉴定PvoBBM,克隆并构建表达载体。使用RT-qPCR分析该基因的表达模式,同时将该基因转入拟南芥植株和本氏烟草(Nicotiana benthamiana)的毛状根中进行功能研究。星油藤AIL基因家族有11个成员,系统进化分析显示其中一个成员xingyouteng_10009416.1与AtBBM亲缘关系最近,其编码蛋白保守基序的种类和数量基本相同,氨基酸序列的相似度为43.7%,命名为PvoBBM。PvoBBM主要在星油藤幼果和聚伞花序顶部表达。过表达PvoBBM可以促进转基因拟南芥生长点以及下胚轴-根过渡区脱分化,少量转基因植株在不含激素的培养基中自发形成再生芽,但在T0代和T1代群体中所占比例较低。以本氏烟草叶片为转化材料,通过发根农杆菌K599介导的遗传转化获得大量转基因毛状根,这些毛状根未发生脱分化,但在其离体培养的叶片上诱导出了转基因再生芽。本研究从星油藤中克隆到具有促进芽再生功能的形态发生基因PvoBBM,为创建星油藤遗传转化体系提供了一个优质的候选基因。

Plukenetia volubilis L.is one of the promising woody oil crops,but its complete and effective genetic transformation system has not been established,which seriously restricts the application process of molecular breeding technology of P.volubilis.BABY BOOM(BBM)is a transcription factor widespread among almost all plants,and is believed to play an important regulatory role in plant embryonic development and plant regeneration.In this study,PvoBBM,a homologs of AtBBM in Arabidopsis thaliana,was cloned from Plukenetia volubilis,and its expression characteristics and functions were studied,which laid the foundation of using this gene to promote transgenic breeding of P.volubilis.The members of AINTEGUMENTA-LIKE(AIL)family were screened from the genome sequence of P.volubilis by local BLAST,PvoBBM was identified by multiple sequence alignments and phylogenetic analysis,and this gene was cloned and constructed into the expression vector.The expression pattern of PvoBBM was analyzed by using RT-qPCR,and this gene was transferred into Arabidopsis thaliana and Nicotiana benthamiana hairy roots for functional studies.There were 11 members of AIL genes family in Plukenetia volubilis,phylogenetic analysis showed that the member xingyouteng_10009416.1 had the closest relationship with AtBBM,the types and numbers of conserved motifs of their encoded proteins were basically the same,and the similarity of their amino acid sequences was 43.7%,which was named PvoBBM.It was showed that PvoBBM was highly expressed in the young fruits and top of cymes of P.volubilis.Overexpression of PvoBBM could promote the dedifferentiation of growing point and hypocotyl-root transition zone of transgenic Arabidopsis thaliana,and a small number of transgenic plants spontaneously formed regeneration buds in hormone-free medium,but the proportion in T0 and T1 generations was low.A large number of transgenic hairy roots were obtained by Agrobacterium rhizogenes K599 mediated genetic transformation using Nicotiana benthamiana leaves as transformation materials.No dedifferentiation was found in the obtained transgenic hairy roots,but transgenic regenerated buds were induced on the leaves cultured in vitro.In this study,PvoBBM,a morphogenetic gene with the function of promoting shoot regeneration,was cloned from Plukenetia volubilis,which provided a high-quality candidate gene for establishing the genetic transformation system of P.volubilis.