摘要
从荔枝‘桂味’的败育胚中提取总RNA,利用GeneRacerTMKit将RNA经去磷酸化反应、去mRNA的帽子结构及RNA Oligo连接,反转录合成cDNA,以cDNA为模板,分别用SSH分离得到的胚败育相关差异表达S-腺苷甲硫氨酸合成酶基因的部分cDNA序列(599 bp)设计出3’和5’端基因特异引物,进行PCR末端扩增。荔枝败育胚的S-腺苷甲硫氨酸合成酶基因序列全长为1515 bp,编码393个氨基酸,与其它物种的S-腺苷甲硫氨酸合成酶基因的核苷酸序列同源性在79％-82％之间,氨基酸序列同源性在88％-91％之间。结果表明所获得的基因为与荔枝胚败育相关的S-腺苷甲硫氨酸合成酶基因。
Total RNA was extracted from aborted-embryo of litchi ' Guiwei'. Dephosphorylating RNA, removing the mRNA cap structure and ligating the RNA Oligo to decapped mRNA with GeneRacer?Kit and then the first strand of cDNA was synthesized by reverse transcription. The 3' and 5' gene specific primers from differential expressing cDNA fragments (SAM gene) , isolated from aborted-embryo of litchi varieties of Guiwei by SSH, was designed with Gene Tools. The full-length 3' and 5' ends of cDNA was amplified with the first cDNA as template and two gene specific primers or nested primers by PCR. Complete SAM gene sequence was obtained by BLAST comparison of the three fragments and splicing according to the overlapping regions. The sequence of the SAM synthetase gene is 1515 bp in length with an opening reading frame (ORF) encoding 393 amino acids. The nucleotide sequence exhibits about 79% -82% overall similarity to the corresponding gene of other organisms, and the amino acids is about 88% -91%. It was suggested that the obtained cDNA was a SAM gene in aborted-embryo of litchi varieties of Guiwei.
出处
《园艺学报》
CAS
CSCD
北大核心
2004年第2期160-164,共5页
Acta Horticulturae Sinica
基金
广东省自然科学基金资助项目(010118)
