建鲤生长激素促泌素受体2（GHSR2s）基因序列、转录变体和GHSRs表达分析

摘要

Growth hormone secretagogue receptors ( GHSRs ) , the endogenous receptor of growth hormone se-cretion ( ghrelin) ,belong to G-protein-coupled receptors family ,and play a regulatory role on animal growth ,energy homeostasis etc.In this experiment,RT-PCR and PCR methods were applied to isolate two jlGHSR2s,jlGHSR2a and 2b,eccoding 366 and 367 amino acids sharing 98% identity with each other,and 95% identity with Danio rerio GHSR2.Intron of jlGHSR2s is located at 793 bp after start codon,there are obvious differences in length and se-quences between 2a and 2b introns.We first report jlGHSR2a′,the variant of jlGHSR2a,which is selectively spliced last 57 bp of exon 1 and reserved last 28 bp of intron near exon 2.jlGHSR2a′encodes 350 amnio acids,missing the last two transmembrane region ,and two ends of splicing sequence are CT-AC.Fluorescent quantitative RT-PCR re-sults showed that the highest expression level of jlGHSRs is in brain and testis ,1s is expressed in all the tissues ex-amined in this experiment ,but2 s is rarely expressed in other tissues .The expression of variants 1 s′and 2 a′has cer-tain degree of positive correlation with 1s and 2s,but 1s/1s′is inconsistent among these tissues ,the highest ratio is Growth hormone secretagogue receptors ( GHSRs ) , the endogenous receptor of growth hormone se-cretion ( ghrelin) ,belong to G-protein-coupled receptors family ,and play a regulatory role on animal growth ,energy homeostasis etc.In this experiment,RT-PCR and PCR methods were applied to isolate two jlGHSR2s,jlGHSR2a and 2b,eccoding 366 and 367 amino acids sharing 98% identity with each other,and 95% identity with Danio rerio GHSR2.Intron of jlGHSR2s is located at 793 bp after start codon,there are obvious differences in length and se-quences between 2a and 2b introns.We first report jlGHSR2a′,the variant of jlGHSR2a,which is selectively spliced last 57 bp of exon 1 and reserved last 28 bp of intron near exon 2.jlGHSR2a′encodes 350 amnio acids,missing the last two transmembrane region ,and two ends of splicing sequence are CT-AC.Fluorescent quantitative RT-PCR re-sults showed that the highest expression level of jlGHSRs is in brain and testis ,1s is expressed in all the tissues ex-amined in this experiment ,but2 s is rarely expressed in other tissues .The expression of variants 1 s′and 2 a′has cer-tain degree of positive correlation with 1s and 2s,but 1s/1s′is inconsistent among these tissues ,the highest ratio is in the liver,followed by muscle,intestine and brain,testis and ovary are the minimum ones .Growth does no signifi-cant effect on the jlGHS R1s expression in brain,but jlGHSR1s expression in liver and intestine of slow growth is higher than fast growth ones, 1s/1s′is also increased obviously.It is opposite in muscle,1s/1s′in male muscle is obviously decreased.The growth speed or starvation could do no significantly effects on jlGHSR2 s expression in the brain.Our results show that the jlGHSR1s expression level is clearly correlated with growth .%应用RT-PCR及PCR分离到鲤鱼2个jlGHSR2s基因，jlGHSR2a和2b，分别编码366，367个氨基酸，相似性高达98％，和金鱼、斑马鱼GHSR2的相似性高达95％。 jlGHSR2s基因的内含子位于起始密码子793 bp后，2a和2b内含子的长度和序列均存在明显差异。试验首次发现了jlGHSR2 a的转录变体（Variant）jlGHSR2a′，由选择性剪切靠近内含子的57 bp外显子1和保留靠近外显子2的28 bp内含子构成，编码350个氨基酸，缺失最后2个跨膜区，剪切序列两端碱基为CT-AC。荧光定量RT-PCR结果表明，jlGHSRs在脑和精巢表达量最高，1s在检测的组织均有表达，但2s在其他组织表达量很少。 jlGHSRs的转录变体1s′和2a′的表达量与1s和2s有一定的正相关性，但1s/1s′在各组织不一致，比值最高出现在肝脏，其次是肌肉、肠和脑，精巢和卵巢最低。生长对脑中jlGHS-R1s的表达量没有显著影响，但生长慢的个体肝和肠中jlGHSR1s的表达量比生长快的高，1s/1s′也有明显升高；肌肉中则相反，1s/1s′在雄鱼肌肉中减小明显。饥饿使雌鱼脑、雌鱼前肠和雌雄鱼后肠jlGHSR1s的表达量显著降低，对其余组织中的表达则不显著。生长快慢或饥饿对脑中jlGHSR2s的表达量均没有显著影响。结果表明，jlGHSR1s的表达量与生长有明显的相关性。