低能离子注入介导的酵母菌对外源植物基因内含子的剪接机制

The existing studies have proved that after the genes including introns from higher plants were transformed into Saccharomyces cerevisiae using traditional transformation methods，they cannot be correctly spliced so that they were not effectively expressed. However, our previous studies show that the genome DNA from the medicinal plant was randomly transformed into Saccharomyces cerevisiae and Hansenula polymorpha by low energy ion implantation, ephedrine-producing and gentiopicroside-producing recombinant yeasts were obtained by mean of the screening methods, respectively. These results indicate that the medicinal plant genes were correctly spliced in the recombinant yeasts, thus realizing the effective expression of the medicinal plant genes. This may be attributed to the genome-wide mutations in recombinant yeasts caused by the interaction between low energy ion beam and the organism including energy deposition, momentum transferring, mass deposition and charge exchange. Then, trans-acting factors of the recombinant yeast can bind efficiently cis-acting elements of medicinal plant genes, thus realizing the splicing of exogenous genes. In this project，in order to analyze the splicing mechanism,the yeast whole-genome re-sequencing technology will be used to investigate the whole-genome-mutation characteristics of the recombinant yeast. The RNA-seq sequencing,yeast one-hybrid,gene deletion and overexpression techniques will be explored to excavate pivotal cis-acting elements and key trans-acting factors that are key to the correct splicing, thus clarifying the splicing mechanism of ephedrine and gentiopicroside biosynthetic gene in recombinant yeasts. This project will provide a sound theoretical basis for the directional modification of the recombinant yeast and offer an alternative strategy for synthetic biology mediated by low energy ion implantation.

现有研究证明利用传统转化方法将含有内含子的高等植物基因导入酿酒酵母中，其内含子不能被正确剪接，基因不能有效表达。我们利用低能离子注入介导药用植物基因组在酿酒酵母和汉逊酵母中转化，分别获得了产麻黄碱和龙胆苦苷的重组菌，结果表明含有内含子的药用植物基因在酵母中发生了正确的剪接，实现了有效表达。这是由于低能注入的离子与酵母菌的相互作用而引发其全基因组范围内的突变，致使重组菌获得了能与顺式作用元件的结构相匹配的反式作用因子，从而实现了对异源基因的剪接。为了解析其剪接机制，拟利用基因组重测序、RNA-seq测序、酵母单杂交和基因缺失/过表达等技术，从全基因组水平上解析重组菌的基因突变特点，挖掘有效剪接所需的主要顺式作用元件和关键反式作用因子，阐明麻黄碱和龙胆苦苷生物合成基因在重组菌中的剪接机制，为重组菌的定向改造和底盘酵母细胞的优化提供理论依据，为基于低能离子注入介导的合成生物学研究提供新思路。

前期研究结果表明，低能离子注入介导含有内含子的药用植物基因在酵母菌中发生了正确的剪接，实现了有效表达。本研究综合运用基因组学、转录组学、代谢组学和生物信息学方法，从全基因组水平上认识离子注入介导的离子束重组酵母菌的基因突变特点和差异表达基因(DEGs)功能，挖掘内含子的有效剪接方式和转录因子种类。基因组学研究结果表明，离子束重组酵母菌基因组中SNP数量为20,409个、有17,802个位点发生了碱基插入或缺失、Contig减少了4条、编码基因减少了38个、非编码RNA数目减少了26个，新增了6种酶类，并在代谢方面与原始菌株表现出了明显差异。DEGs的GO功能研究结果显示，离子束重组酵母菌新增了5个生物学过程功能、2个细胞组分功能和7个分子功能。DEGs的KEGG代谢通路分析表明，离子束重组酵母菌新增了16条代谢通路，其中包括生物碱、萜类等次生代谢产物生物合成必须的酪氨酸代谢通路、苯丙烷类生物合成通路和甲羟戊酸代谢通路，相关DEGs调控的关键小分子标记物的合成速率与代谢组学方法测得的相关产物产量的变化趋势一致。在功能基因的可变剪接研究中，发现了离子束重组酵母菌的5607个基因发生了81种类型的可变剪切，其中外显子跳跃(SE)73种、3'端可变剪切(A3SS)5种、外显子互斥(MXE)3种。与原始菌株相比，离子束重组酵母菌新增了A3SS和SE剪接类型各1种，缺失5'端可变剪切(A5SS)和内含子保留(RI)可变剪接类型。对SE的进一步研究发现，离子束重组酵母菌发生SE可变剪切的基因归类于4个Motif，并注释获得了34个转录因子，其中特别重要的是，两类AP2/ERF domain转录因子(AT1G77640和AT4G31060)为离子束重组酵母菌新增的来源于植物的转录因子。这说明在离子注入介导外源药用植物基因组DNA随机转化酵母菌的过程中，不仅是与植物天然产物生物合成相关的功能基因被整合至酵母菌基因组，而且相关的转录因子的基因也转入酵母菌，且能正确转录和表达。本研究结果丰富了低能离子注入介导远缘、超远缘物种间遗传物质交流的理论体系，对人们深入理解低能离子注入介导外源DNA大分子遗传转化的分子机制具有重要的科学意义。