玉米光周期敏感类Hd6基因的克隆和实时定量表达分析

水稻Hd6基因在调节水稻光周期敏感中起重要作用。本研究以热带玉米自交系CML288为材料, 利用同源基因克隆法结合3¢RACE技术克隆了与水稻Hd6基因同源的玉米类Hd6基因。该基因序列中999 bp的开放阅读框可编码332个氨基酸。BLAST分析发现, 该基因与玉米中编码蛋白激酶CK2的一个基因高度同源, 所推测的氨基酸序列包含2个CK2活性区域, 1个N末端区域, 1个ATP结合位点和1个丝氨酸/ 苏氨酸蛋白质激酶活性位点,与玉米、小麦、水稻和拟南芥的CK2氨基酸序列也有较高的同源性。建立了SYBR GREEN法实时荧光定量RT-PCR表达分析系统, 研究了不同光周期处理下类Hd6基因在光周期敏感自交系CML288茎尖和叶片中的表达。结果发现, 该基因在茎尖和叶片中均有表达。在短日条件下, 该基因在6~8叶期茎尖中的表达量存在明显差异, 在光周期敏感的7叶期出现表达高峰, 在叶片中的表达量均低于茎尖。在长日条件下, 该基因在茎尖中6叶期表达量较低, 在光周期敏感的9叶期在叶片中高量表达。由此可推测, 该基因与玉米光周期敏感密切相关, 可能在光周期敏感调节过程中发挥一定的作用。

Clone and Quantitative Analysis by Real-Time RT-PCR of Photoperiod Sensitive Gene Hd6-Like in Maize

Hd6 gene plays an important role in photoperiod sensitive phenomenon in rice (Oryza sativa L.). In the present study, a Hd6-like gene was cloned by RT-PCR and rapid amplification of cDNA ends (RACE) using a photosensitive tropical maize (Zea mays L.) inbred line CML288. A predicated 999-bp open reading frame was detected in the cloned sequence, which encoded 332 amino acids. By BLAST analysis, we found the gene sequence shared a high similarity with a gene encoding protein kinase CK2 in maize. The amino acid sequence contained two CK2 activity domains, one ATP binding site, one basic stretch (NLS), one N terminal domain, a putative serine/thereonine protein kinase active site, and showed a high homologization with that of protein kinase CK2α in maize, wheat, rice and Arabidopsis. We established fluorescence quantitative RT-PCR system with CML288, and studied the expression of Hd6-like in shoot apical meristem (SAM) and leaf under long day and short day conditions between 5-leaf and 9-leaf stages. We found that Hd6-like gene expressed both in SAM and leaf of CML288. Under the short day treatment, it expressed higher in SAM than in leaf from 6-leaf to 8-leaf stages with the highest expression level at 7-leaf stage. Under the long day treatment, the expression level fell down suddenly at 6-leaf stage in SAM with the peak at 9-leaf stage in leaf. Our results suggest that Hd6-like gene encodes protein kinase CK2α that closely relates to photoperiod-sensitive, and may function in regulating photoperiod sensitivity in maize.