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克隆分离了长石莼(缘管浒苔)光合作用第一关键酶Rubisco大亚基全长基因(rbcL)。首先应用PCR和RT-PCR方法扩增rbcL大片段DNA序列和大片段cDNA序列,结果表明,获得的2个克隆序列完全相同,该rbcL大片段基因不存在内含子。其次应用基因步移方法分别对rbcL基因的5′上游未知序列和3′下游未知序列进行扩增,在此基础上,将3个片段序列进行拼接,获得了rbcL全长基因序列(NCBI登陆号:DQ813497)。序列全长2124bp,其中编码区序列长1425bp,为单外显子基因,编码474个氨基酸;5′非翻译区序列长225bp,转录起始位点为位于翻译起始密码子ATG上游第47个核苷酸G,在距离转录起始位点-9bp—-48bp的范围内有推测的类似原核生物的启动子序列(-10区:TAAAAT、-35区:TTGAAA);3′非翻译区序列长474bp,在距离转译终止密码子TAA下游54—98bp处有一段较长的回文序列,可形成一个22bp的茎结构。密码子偏好性分析结果表明,长石莼(缘管浒苔)rbcL基因偏向使用第三位核苷酸碱基为A和T的密码子。
The Rubisco large subunit full-length gene (rbcL), the first key enzyme in photosynthesis of Phyllosticta furbaura was isolated by cloning. First, PCR and RT-PCR methods amplified rbcL large fragment DNA sequence and large fragment cDNA sequence, the results showed that the two cloned sequences were identical, the rbcL large fragment gene does not exist introns. Secondly, the 5 ’upstream unknown sequences and 3’ downstream unknown sequences of rbcL gene were amplified by gene walking method respectively. Based on these, the 3 fragments were spliced to obtain the full-length rbcL gene sequence (NCBI accession number : DQ813497). The full length of the sequence was 2124bp, in which the coding region was 1425bp in length, which was a single exon gene encoding 474 amino acids. The 5 ’untranslated region was 225bp in length and the transcription initiation site was located at the 47th upstream of the translation initiation codon ATG Nucleotide G, a putative prokaryotic similar prokaryotic promoter sequence (-10 region: TAAAAT, -35 region: TTGAAA) within the range of -9 bp to 48 bp from the transcription start site; 3 ’untranslated region sequence It is 474bp long and has a longer palindromic sequence 54-98bp downstream of the translational stop codon TAA to form a 22bp stem structure. The results of codon bias analysis indicated that the rbcL gene of Phyllosticta spp. Biased toward the third codon using nucleotide bases A and T.