为了克隆多穗柯4-香豆酸辅酶A连接酶(4-coumarate-CoA ligase,4CL)基因,了解其表达情况,从而为深入研究4CL基因分子功能及黄酮类化合物生物合成奠定基础,依据多穗柯转录组测序结果,设计特异性引物,PCR扩增得到4CL基因cDNA全长序列,利用相关软件进行生物信息学分析;采用qRT-PCR法检测4CL基因在不同器官中的表达情况,使用SPSS18.0软件分析其表达量与根皮苷含量间的关系.结果表明:多穗柯4CL基因cDNA全长1704 bp,包含长1629 bp的开放阅读框,编码含542个氨基酸的蛋白质.该蛋白定位于细胞质中,属亲水性蛋白.多穗柯4CL基因在除根以外的各个器官中均有表达,且表达量与根皮苷含量间呈极显著的正相关关系(P<0.01).

利用生物信息学方法比较壳斗科6个属14个物种的叶绿体基因组间差异,以近缘物种榛为外类群构建系统进化树,揭示壳斗科叶绿体基因组的结构特征及变异规律。结果显示,14种壳斗科植物的叶绿体基因组均为双链环状分子结构,大小在160 kB左右,差异较小,最大仅差1 366 bp;基因顺序基本一致,而基因数量有所差异,infA、petG、rpl22、ycf1、ycf15等多个基因在部分物种中发生丢失;主要有32个蛋白编码基因长度发生变异,其原因是内含子的丢失、内含子或者编码区的长度改变,华南锥基因长度变异较大; 4个IR边界相对保守,但锥栗、Castanea pumila、华南锥3个物种由于边界扩张导致rps19基因部分序列进入到IR区;以榛为外类群构建的系统发育树,各进化支支持率较高,分辨率较好。研究结果表明,叶绿体基因组可以用于分析关系较近与进化较快物种的系统发生问题,为系统发育和进化研究提供依据。

Objective To understand the chalcone isomerase (CHI) gene expression by cloning it in Lithocarpus polystachyus.Methods A full-length cDNA of CHI gene from Lithocarpus polystachyus (Lpr-CHI) was obtained by PCR cloning technique according transcriptomics sequences infromation,which bioinformatics analysis was carried out.The expression of CHI gene in different organs ofLithocarpus polystachyus was detected by qRT-PCR.Results Lpr-CHI was 772 bp in full length with an open reading frame (ORF) of 696 bp,which encoded a protein with 231 amino acids.The protein did not contain a transmembrane domain and is localized in the cytoplasm.Lpr-CHI gene expression was found in different parts,and reached the highest in leaf,which was 9.75 times of the least gene expression in root.Conclusion Lpr-CHIwas obtained for the first time,and it was clear that the gene belongs to CHItype Ⅱ.And the expression of Lpr-CHI in each organ was significantly different.

Objective To clone flavanone 3-hydroxylase (F3H) gene from Lithocarpus polystachyus,and to understand its gene characteristics and initially investigate its expression level in different organs.Methods The total RNA and genomic DNA from blade of L.polystachyus were extracted.Based on the result of RNA-seq,a pair of specific primers were designed.cDNA and DNA sequences of F3H gene from L.polystachyus were amplified by PCR,then bioinformation analysis was performed after sequencing.The expression level of F3H gene in different organs ofL.polystachyus was detected by qRT-PCR.Results The full length of cDNA of F3H gene was 1 340 bp containing a 1 092 bp open reading frame that encoded 393 amino acids,and F3H was located in the cytoplasm.The result of qRT-PCR showed that F3H gene expressed in different organs ofL.polystachyus,and the expression levels of F3H gene were significantly different in different organs (P ＜ 0.05).Conclusion The F3H gene ofL.polystachyus was cloned and its bioinformation was analyzed for the first time,proving that the expression level ofF3H gene in different organs ofL.polystachyus was different.This finding lays a foundation for the studies on secondary metabolism of flavonoids in L.polystachyus.