海巴戟果实软化相关基因McXTH的克隆和表达分析

【目的】了解诺丽果实软化规律,克隆木葡聚糖内转糖苷酶/水解酶(XTH)基因,初步明确其表达模式。【方法】用果实硬度计测量后熟阶段的诺丽果实在25℃放置及乙烯利处理两种状态下的硬度变化。从诺丽果实采后0~48 h转录组数据中筛选差异表达的XTH候选基因。通过生物信息学分析,克隆目的基因全长,并利用荧光定量PCR技术分析基因表达模式,同时用便携式乙烯测量仪测定内源乙烯释放量。【结果】后熟阶段的海巴戟果实采后表皮硬度随贮藏时间增加而下降,且乙烯利处理后的软化趋势较室温自然放置更为显著。从海巴戟果实采后0~48 h转录组数据中筛选出基因XTH(DN16278g1)的表达量高、差异大。该基因具有完整的开放阅读框(ORF),克隆测序后将其命名为McXTH,并将其提交Genbank获得登录号为ON512442。McXTH基因ORF全长1062 bp,共编码353个氨基酸,与其他植物中的XTH基因氨基酸序列的相似性为75%~88%。RT-qPCR结果和内源乙烯释放量表明,在海巴戟果实成熟阶段McXTH的表达量与同时期的内源乙烯释放量在趋势上大体相同。【结论】海巴戟果实的软化受乙烯和关键基因XTH调控,Mc XTH的克隆为进一步进行XTH基因在海巴戟果实成熟软化过程的功能研究夯实基础。

木葡聚糖内糖基转移酶/水解酶(XTH)研究进展

木葡聚糖(XG)作为双子叶植物初生细胞壁中最主要的半纤维素,一直是当代植物细胞模型研究中持续关注的焦点。木葡聚糖内糖基转移酶/水解酶(xyloglucan endotransglucosylase/hydrolase,XTH)能够催化木葡聚糖分子水解或转移,对细胞壁木葡聚糖主链实现剪切或重排,在植物细胞壁伸展性方面发挥重要作用。本文就酶的结构基础、作用机制及在植物生长发育中的作用等方面进行综述,以期为后续的XTH基因产物的功能研究提供相关证据。

大丽花花瓣生长与衰老相关基因DpXTH的表达分析

为了研究木葡聚糖内糖基转移酶/水解酶(XTH)基因在大丽花花瓣生长和衰老过程中的功能,本试验以大丽花(Dahlia pinnata Cav)花瓣为材料,在克隆大丽花花瓣XTH基因cDNA序列(DpXTH)的基础上,以18S rRNA为内参基因,利用半定量RT-PCR技术,检测了大丽花花瓣生长与衰老过程中DpXTH基因的表达变化。结果显示,大丽花开花过程中DpXTH的表达量急剧增加,盛花期达到最大,而衰老期又迅速下降,表明DpXTH与花瓣的生长发育密切相关,可能在大丽花花瓣生长的不同阶段DpXTH对细胞壁中木葡聚糖的代谢具有不同的调控方式。

Identification of Senescence-associated Protein DpXTH1 and its Gene Cloning in Dahlia Petals

[Objective] This study aimed to explore the molecular mechanism of senescence in ethylene-insensitive flowers. [Method] The dahlia petals were used as matedal, and the senescence-associated proteins were isolated and identified using two-dimensional electrophoresis, mass spectrometry and an encoding gene was cloned using molecular biology techniques. [Result] In the two-dimensional elec- trophorogram of proteins from dahlia petals at building color, full flowering and flow- er senescence pedods, a total of 44 protein spots with differences in expression level more than two times were detected. From the 44 protein spots, xyloglucan （XTHs）, a senescence-associated protein, was iso- lated and identified and its expression level was increased continuously with the senescence process of dahlia petals. By using the total RNA of dahlia petals as matedal and a pair of degenerate pdmers, the cDNA sequence of XTH gene was cloned by RT-PCR. The encoding region of XTH gene has a full length of 882 bp, encoding 293 amino acid residues, and is named as DpXTH1 （Accession number： HM053613.1）. The cluster analysis showed that the amino acid sequence of DpXTH1 has high homology with those of XTHs in other plants. [Conclusion] The isolated and identified DpXTH1 from dahlia petals belonged to the XTH family in plants, and its biological function was associated with the senescence process and regulation of dahlia petals.

Analysis on Tissue-specific Expression of Dp XTH1 and Dp XTH2 Genes in Dahlia

[Objective] This study aimed to investigate the functions and related mechanisms of xyloglucan Endotransglycosylase/hydrolases （XTHs） during the growth and development of dahlia. [Method] Using /3-actin as the reference gene, the rela- tive transcription levels of DpXTH1 and DpXTH2 genes in roots, stems, leaves and petals of dahlia were analyzed by real-time RT-PCR. [Result] The DpXTH1 and DpXTH2 were not expressed in the roots, but expressed abundantly in the petals of dahlia. There were little expressions in the stems and leaves of dahlia. [Conclusion] The DpXTH1 and DpXTH2 were petal-specific genes and closely related to the growth and development of petals in dahlia.

拟南芥XTH33基因的CRISPR/Cas9高效敲除质粒构建

为解决XTH33基因高效敲除质粒的构建难题,本文对拟南芥CRISPR/Cas9高效敲除质粒的gRNA靶点进行研究,提出靶向拟南芥XTH33基因的gRNA经体外活性检测,可初步筛选高效高活性的gRNA,3条靶向拟南芥XTH33基因的gRNA,XTH33-G3敲除活性最高,达到50%以上,将XTH33-G3靶点插入质粒骨架pUbi-atU6-gRNA,经测序鉴定成功构建载体pUbi-atU6-gRNA-XTH33。以期为进一步进行XTH33基因功能鉴定奠定基础。

甘蓝型油菜BnXTH1基因的克隆与表达分析

木葡聚糖内糖基转移酶/水解酶(XTH)是一类庞大的多基因家族,在细胞壁形成以及植物生长发育过程中起重要作用。本研究利用同源序列设计引物进行PCR扩增,克隆得到甘蓝型油菜XTH基因的cDNA序列,将其命名为BnXTH1(GenBank登录号:KF612342)。BnXTH1的开放阅读框长855 bp,编码284个氨基酸,预测的蛋白质相对分子质量为32.46 kD,等电点为8.84,含有典型的XTH蛋白催化活性序列DEIDFEFLGN。系统进化分析表明该基因与拟南芥XTH17进化关系最近。半定量RT-PCR分析结果显示,BnXTH1在花和根中表达量较高,其在幼苗中的表达受赤霉素、油菜素内酯以及生长素的诱导,推测该基因可能与幼苗生长发育的调控有关。