棉铃虫性染色体两种分子标记的克隆及序列分析

Cloning and sequencing of two molecular markers located on the sex chromosomes of the cotton bollworm, Helicoverpa armigera

为了建立棉铃虫Helicoverpa armigera性染色体的特异性分子标记,利用RAPD-PCR技术对雌雄棉铃虫基因组DNA进行筛选,从500种随机引物中筛选到1条引物(Operon编号为AF-18) ,可扩增出1条约450 bp的雌性特异片段。经克隆测序并合成特异引物进行验证,表明该片段为棉铃虫雌性特异分子标记,位于W染色体上。利用家蚕、果蝇等昆虫Kettin基因序列,克隆了棉铃虫的同源基因HaKettin片段,并采用荧光定量PCR技术,以棉铃虫的DH-PBAN基因为参照基因,检测棉铃虫雌雄不同个体间HaKettin基因与DH-PBAN基因的拷贝数之比,结果表明:雄体HaKettin∶DH-PBAN=1.0 ,雌体HaKettin∶DH-PBAN=0.5 ,据此推断HaKettin基因位于棉铃虫Z染色体上。

The individual genome DNA of male and female cotton bollworms （Helicoverpa armigera） was screened with RAPD-PCR technology by 500 random primers. A 450 bp marker amplified from female genome DNA with random primer Operon AF-18 was cloned and sequenced, which was confirmed as a specific molecular marker on the W chromosome. A part of HaKettin gene was cloned according to the sequences of homologues from Bombyx mori and Drosophila, and its copy number in the genome DNA of male and female cotton bollworms was detected with the real-time quantitative PCR technique compared with DH-PBAN. The ratio of copy between HaKettin and DH-PBAN was 1.0 in males and 0.5 in females, showing that the copy number of HaKettin is equal to that of Z chromosome in the genome of cotton bollworm. It is so inferred that HaKettin is located on Z chromosome in the genome of cotton bollworm.