鸡瘦蛋白基因克隆中的若干问题研究

Issues on the cloning of chicken Leptin gene

根据GenBank中鸡的LeptinmRNA序列(AccessionNo.AF012727)设计12对引物,用RT-PCR方法从不同品种(系)、不同时期及不同处理鸡的脂肪、肝脏和卵巢组织总RNA中没有扩增出鸡的Leptin基因片段;根据鸡的EST数据库中查到的一条鸡Leptin基因前体序列设计4对引物,用RT-PCR方法在包括卵巢在内的多个组织的cDNA中没能扩增出正确序列。为提高鸡Leptin基因的表达水平,通过给鸡注射胰岛素,利用RT-PCR方法对肝脏、卵巢和脂肪组织的总RNA进行扩增,没有得到目的序列。根据已发表的哺乳动物的Leptin基因序列设计兼并引物对鸡基因组DNA和脂肪、肝脏组织的cDNA进行PCR,结果没有特异性扩增条带,但在小鼠的基因组中可以获得稳定的扩增条带。用扩增长片段LATaq酶从鸡基因组DNA中也没有扩增出Leptin基因片段,而从小鼠的基因组DNA中,可扩增出小鼠Leptin基因片段;以小鼠LeptincDNA片段为探针,对鸡脂肪组织和肝脏组织来源的总RNA进行NorthernBlot分析,并未获得杂交信号;以猪的LeptincDNA片段为探针,对鸡基因组DNA进行SouthernBlot分析,并未获得特异性的结果。研究结果表明,在鸡的脂肪、肝脏和卵巢组织中不存在与小鼠Leptin基因同源性如此高的mRNA序列,在鸡的基因组中也不存在与小鼠、猪等哺乳动物Leptin基因序列同源性如此高的基因?

Twelve pairs of PCR primers based on the chicken Leptin mRNA sequenc e in Genbank (Accession NO. AF012727) were designed to amplify the gene. PCR amp lification was carried out on reverse-transcribed mRNA of the fat, liver, and ov ary from the domestic chickens of different weeks of age from different breeds o r lines. No PCR products sharing close similarity to the mouse Leptin sequence w ere generated from any cDNA templates. Another four pairs of PCR primers were de signed according to an EST fragment of chicken Leptin precursor found at the chi cken EST library. RT-PCR method was developed to amplify the RNA templates from various tissues including ovary. No expected PCR products and sequence were gott en from these templates. In order to enhance the copy of Leptin mRNA, the chick en were treated with injecting insulin and RNA templates were collected from liv er, ovary and adipose tissue. Above pairs of PCR primers were used and RT-PCR wa s carried out, but we did not get specific PCR products and sequences. A pair of degenerate PCR primers, based on the sequences of reported mammal′s Leptin gen e, were designed and PCR amplification was carried out on chicken genomic DNA, c DNA from liver and adipose tissues and mouse gonomic DNA templates. We also did not get expected PCR products and sequences from chicken genomic DNA and cDNA te mplates but amplification of mouse Leptin sequence was consistently obtained whe n control mouse gonomic DNA templates were used. Four pairs of specific PCR prim ers were designed and the LA Taq, possessing the ability to amplify long fragmen t, was used to amplify the mouse and chicken genomic DNA templates, we got the p ositive results from mouse not chicken. Northern hybridization using a mouse Lep tin probe failed to produce a signal with RNA from chicken liver and adipose tis sue but a strong signal was obtained from control mouse fat total RNA. Southern hybridization revealed no signal using a pig Leptin probe to chicken genomic DNA . It is concluded that mRNA sharing high sequence identity with mouse Leptin is not exist in the fat, liver or ovary of the domestic chicken,and a chicken Lepti n gene sequence with close sequence similarity to mouse and pig Leptin is not ex ist in the chicken genome.