小麦光周期基因Ppd-B1的选择性剪接分析

Alternative Splicing of Photoperiod Response Gene Ppd-B1 in Wheat

为深入认识光周期基因Ppd-B1的功能,在转录水平上研究其表达特点,通过cDNA与基因组DNA序列对比,发现Ppd-B1mRNA加工存在选择性剪接,3个可选择剪接位点分别以外显子增加、5'剪接位点改变和内含子保留形式分布在5′UTR、第5外显子和第6内含子,其中前两种方式不引起蛋白保守结构域的改变,后种方式却导致基因移码突变。Ppd-B1选择性剪接可产生8种不同形式的转录本,其中4种含有完整的编码序列,能够翻译成功能蛋白,另4种表达丰度较低,不翻译或翻译时被提前终止。Ppd-B1不同转录本的相对表达量不同,而且这种差异受材料光周期反应特性和外界光周期环境的影响。

Alternative splicing creates multiple types of mRNA transcripts from a single gene, and can contribute to the regulation of gene function and protein diversity in eukaryotic cells. Photoperiod response plays a major role in controlling the heading time, yield, and adaptability in wheat （Triticum aestivum L.）, and Ppd-B1 has been considered to have a potential effect on wheat responses to photoperiod changes. However, its aiternative splicing has not been reported yet. In the present study, a photoperiod insensitive variety Chinese Spring carrying Ppd-B1 and another photoperiod sensitive variety Marquis carrying ppd-B1 were grown under four different photoperiod conditions. The cDNA and genomic DNA sequences of Ppd-B1 were isolated and characterized. The length of Ppd-B1 coding region was 3 053 bp with eight exons whose total size was 1 995 bp. The Ppd-B1 mRNA was alternatively spliced, producing multiple types of transcripts. There were three alternative splicing sites located in 5＇ UTR, exon 5, and intron 6, whose action led to exon increasing, alternative 5＇ end processing and intron retention. The alternative processing events that were augmented by the two former splicing sites remained the conserved Psedo-response regulator （PRR） domain in the deduced protein, whereas that directed by the third site resulted in frameshift mutation. Among the eight different types of Ppd-B1 transcripts produced by alternative splicing, four types （type a to d） expressed at relatively high levels with the frequency ranging from 32.6% （type d） to 13.0% （type b）. They could be translated into full length proteins, and were likely to be functional. The remaining types （type e to h） expressed at very low levels with the frequency varying from 2.2% （type g） to 6.5% （type e）. They gave rise to truncated peptides upon conceptual translation. The relative abundance of the different types of Ppd-B1 transcripts were changed by the photoperiod response characteristics of wheat varieties and the photoperiod conditions under which wheat plants were cultured. The data collected here paves the way for further studies to reveal the physiological function of Ppd-B1 alternative splicing in wheat.