中华猕猴桃fasciclin基因家族生物信息学分析及克隆

Bioinformatics analysis and cloning of fasciclin gene family in kiwifruit

【目的】探讨fasciclin基因在中华猕猴桃中的生物信息。【方法】对中华猕猴桃雌株38个fasciclin基因进行生物信息学分析和克隆其中一个与性别相关的fasciclin基因。【结果】38个fasciclin基因编码133~606个氨基酸。丝氨酸、苏氨酸和酪氨酸磷酸化位点范围分别为10~56个、2~26个、0~8个。38个fasciclin基因拥有1~9个motif结构(取值10),其中Motif3为共同具有。68.42%fasciclin蛋白为不稳定疏水性蛋白。2 000 bp启动子区共预测到TFs 110个,较多的TFs依次为BBR-BPC、TALE和AP2,9个基因没有预测到TFs结合位点。UPGMA聚类法把38个fasciclin蛋白分成2类。基因克隆序列(723 bp)具有fasciclin保守结构域,该序列只比对到8号染色体上。【结论】fasciclin基因保守性不强;克隆序列与中华猕猴桃雌雄性状显著关联,但与美味猕猴桃雌雄无关联;雌株中的同源区没有注释到基因。

【Objective】Actinidia chinensis var.chinensis and A.chinensis var.deliciosa are mostly valuable in the production of kiwifruit.Former research showed that male plants used as rootstocks had strong grafting advantages.Most of the rootstocks currently used in the production are 1-2 year old seedlings.Both female and male plants are used as rootstocks due to the lack of efficient method to distinguish the gender of the young seedlings,leading to the inconsistent growth and fruit quality of kiwifruit Therefore,it is very important to identify the gender of kiwifruit at the seedling stage.It was reported that the fasciclin genes were associated with the gender and participated in plant growth and pollen fertility development.The work was designed to study the biological information of fasciclin gene family in A.chinensis var.chinensis to provide theoretical basis for exploring the role of fasciclin genes in the differentiation of the male and female plants of kiwifruit and developing markers for identifying the male and female plants at seedlings stage.【Methods】The gene structure characteristics,evolutionary relationships,protein characteristics,promoter prediction,secondary structure of 38 fasciclin genes from the female plant of A.chinensis var.chinensis were analyzed by bioinformatics software A gender-related fasciclin gene,named FrBy,was cloned from the male and female plants of A.chinensis var.chinensis and A.chinensis var.deliciosa,respectively.and the FrBy gene sequence was analyzed using existing knowledge and affordable tools,including reference genome of the female plant named Red 5 of A.chinensis var.chinensis.【Results】The 38 fasciclin genes were distributed on the remaining 18 chromosomes except for 1,4,5,6,7,10,11,15,16,19 and 21 chromosomes.The genes encoded 133-606 amino acids in length,and the number of serine phosphorylation sites,threonine phosphorylation sites and tyrosine phosphorylation sites ranged from 10 to 563 to 26 and 0 to 8,respectively.The majority of serine phosphorylation sites were found in PSS24547.1 protein,the fasciclin protein on chromosome 8.A total of 110 transcription factors(TFs)were predicted in the 2000 bp promoter region.The first three TFs were BBR-BPC,TALE and AP2.They were all related to the sex development of plants.The binding sites of the TFs were not predicted from nine fasciclin genes,in which gene silencing might occur during evolution.The maximum motif structures were 9 and the minimum was 1 of the 38 fasciclin genes.The PSS11741.1,PS01642.1 and PSS24547.1 proteins were choosenas the most representive characteristic sequences to set up the homologous tertiary structure model.The tertiary structure analysis showed that the PSS016442.1 mostly contained theα-helices.The PSS24547.1 mostly contained theβ-folds and random curls The PSS11741.1 least contained theβ-folds and random curls.About 68.42 percent of 38 fasciclin proteins were hydrophobic proteinw which were unstable.The 38 fasciclin proteins were clearly divided into two subgroups by UPGMA(unweighted pair-group method with arithmetic means)method,among them,the PSS24547.1 was in its own class.The FrBy gene with 723 bp sequence that cloned from the male plant possessed a Fas conserved domain,the 723 bp sequence was only aligned with a region on chromosome 8 of the reference genome of Red 5(E-value is 5 E-167).【Conclusion】The study showed that the fasciclin genes were not conservitive in the female plant of A.chinensis var.chinensis while the FrBy gene cloned from the male plant of A.chinensis var.chinensis was very conservitive.The 723 bp sequence was significantly related to the gender character of A.chinensis var.chinensis.However the 723 bp sequence could not distinguish the gender of Actinidia chinensis var.deliciosa,as a functional dioecious plant.The differentiation of varietas might occrue earlier than that of gender in A.chinensis.