Structure And Expression Analysis Of Three Types Of Pyruvate Kinase And α-AMY Gene Of Grass Carp(Ctenopharyngodon Idella) And Screening Of SNPs Markers Related To Growth Traits

Sugar added to feed is a kind of energy substance of low cost. But compared with the sugar utilization of mammals, utilization ratio of fish is low.In this study, three types of pyruvate kinase genes and α- amylase(α-AMY) gene, which are key enzyme genes associated with sugar using, were selected as studying objects. c DNA sequence, tissure distribution and expression pattern in early development stages of these genes in grass carp were analyzed. What’s more, these genes were used as candidate genes to identify the association of SNPs with growth traits in grass carp group in order to indicate the amount of sugar added to feed and provide markers for molecular marker assisted breeding of grass carp. The detailed contents were as follows:1. Analysis of c DNA sequence,tissue distribution and expression in early development of pyruvate kinase gene in grass carp. Analysis on c DNA sequence showed: the PKL ORF was 1617 bp in length and encoded 538 amino acids, the PKMb ORF was 1587 bp and encoded 528 amino acids, PK-like ORF was 1599 bp and encoded 532 amino acids. We performed relative quantitative studies of the expression of PKL, PKMb and PK-like m RNAs in different tissues and early development of grass carp by real time PCR. Relative quantitative analysis on different tissues showed: the PKL gene mainly expressed in liver, foregut, midgut and kidney, PKMb gene expressed highest in skeletal muscle, PK-like gene mainly expressed in heart. The Amino acid sequence homology between grass carp PKL,PKMb and zebrafish liver-type PK,muscle-b-type PK were 90% and 93% respectively which were the highest, and the main distribution tissues and key active sites between them are basically the same. So we can identify that PKL and PKMb are respectively liver-type and muscle-b-type PK isozymes of grass carp. The sequence homology between grass carp PK-like and zebrafish muscle-a-type PK was as high as 95%, but their main expressing tissues are not the same.The identity between PK-like and PKM1 of mammals is the highest, but the key active sites between them are not the same. Therefore which PK isozyme type of PK-like belong to need to be studied further.Relative quantitative analysis on early development showed there is a lot of PKMb and PK-like m RNA in unfertilized eggs, while the PKL began to express from mid-gastrula. From fertilized eggs to 48 hours post hacthing, the expression of PKMb and PK-like were higher than that of PKL; after 72 hours post hacthing, the expression of PKL increased significantly.In oocytes and early embryos,PKMb and PK-like m RNAs occurred abundantly as maternal m RNAs, but PKL m RNAs did not occurred.However,after the larva was fed, PKL expression rise significantly(P < 0.05).2. Polymorohisms of SNPs in PK-like and PKL gene and association analysis with growth traits in grass carp. Three pairs of primers were designed according to the sequences of many available contigs in the EST database of grass carp. Two SNP sites, H3(T+1819C) and H4(G+1820T),were identified with the method of direct sequencing and sequence alignment. Genotype frequency in 3 grass carp group was tested with the method of Sna Pshot and they were found with a complete linkage. The sites of H3 and H4 formed three genotypes(AA, AB, and BB).The correlation between PK-like gene SNPs and growth traits of grass carp in random group analyzed by a general linear model was not significant.But the weight gain of genotype AB was 0.6 % and 3.4% faster than that of AA and BB respectively. For the two group fed with different level of sugar, weight gain of genotype AA was 4.9% and 6.1% faster than that of AB and BB respectively in group fed with normal level of sugar,but the weight among them were not different significantly.While in the group fed with high-level sugar, body weight of genotype AA was significant heavier than that of genotype AB and BB(P<0.05), and difference of body weight between genotype AB and BB were not significant(P>0.05). Weight gain rate of genotype AA was 36.8% and 30.4% faster than that of AB and BB respectively. What’s more, the average body weight gain of genotype AA group fed with high-level sugar was 11.4% faster than that of AA group fed with norml sugar, which was different from genotype AB and BB. Average body weight of genotype AB and BB group fed with normal sugar were heavier than that of AB and BB group fed with high-level of sugar.The results indicat that genotype AA of grass carp grow faster when fed with high-level sugar.It was speculated that genotype AA of grass carp can make better use of sugar added to feed.Three SNP sites, H5(T+561C),H6(T+744C)and H7(A+762G) were identified in PKL, and they were found complete linked with the method of Sna Pshot. The sites of H5 and H7 formed three genotypes(AA, AB, and BB).Weight gain of genotype AA was 6.4% and 9.2% faster than that of AB and BB respectively in random group, while genotype TT of H6 site was 9.0% and 11.0% faster than that of TC and CC in random group. But none of the three SNP sites were significantly related to grouth traits of grass carp in random group(P>0.05). In the two group of grass carp fed with two different level of sugar, both of H5(T+561C)site and H7(A+762G) site show no significant correlation with body weight of grass carp in the two group(P>0.05),but weight gain of genotype AA was 15.7% and 7.2% faster than that of AB and BB respectively in group fed with normal level of sugar, and 17.4% and 11.2% faster than that of AB and BB in group fed with high level of sugar. As for H6(T+744C) of grass carp group fed with normal sugar,body weight of genotype TT group was significantly heavier than that of genotype TC group(P<0.05),and weight gain of genotype TT was 32.0% and 21.2% faster than that of TC and CC. On the other hand, H6 site did not show significant correlation with growth traits in random group of grass carps whose body weight were all heavier than 900 g, while in group whose body weight were all less than 150 g fed with norml sugar it showed significant relation with body weight. It was speculated that the influence SNP had on growth traits of grass carp may be related to the growth stages of grass carp.3. Analysis of c DNA sequence,tissue distribution and expression in early development of α-amylase gene in grass carp. As the sequence analysis showed: the α-amylase gene ORF was 1539 bp in length and encoded 512 amino acids, and the Amino acid sequence homology between grass carp AMY and other species was very high. The grass carp AMY had the highest homology, 92%, with α-amylase of zebrafish(Danio rerio), and had the lowest homology,72%,with human beenings. Relative quantitative expression of α-amylase m RNA in different tissues and different development stages of grass carp were studied by real time PCR. Relative quantitative expression of α-AMY m RNA in different tissues showed: the α-amylase gene expressed most in hepatopancrea, and secondly in gut. As the analysis of relative quantitative expression on grass carp in early development showed: α-AMY m RNA can be tested until at the stage of organogenesis, and was very low till 48 h after hatching. However, after 72 h after hatching, the expression of α-AMY increased obviously. Analyzing the tissue distribution and the expression pattern in early development we could find out that α-amylase could be generated both in hepatopancrea and gut. Expression of α-AMY increased obviously 72 h after hatching when the larvae began to feed. While the amount of m RNA 120 h and 144 h after hatching were 4.5 times and 32.0 times as much as 72 h after hatching respectively. The results may indicate rational amount of sugar added to feed in early development of grass carp.4. Polymorohisms of SNPs in α-amylase(α-AMY) gene and association analysis with growth traits in grass carp. Two SNP sites, H1(T+357A) and H2(T7A),were identified in α-AMY gene. The correlation between α-AMY gene SNPs and growth traits was analyzed by a general linear model. The results show that none of the two SNPs have significant correlation with growth traits of grass carp in random group(P>0.05).But weight gain of genotype AA in H1 site was 0.4% and 1.3% faster than that of genotype AT and TT respectively, and weight gain of genotype of TT in site H2 was 3.1% faster than that of genotype AA. Meanwhile,both of the SNPs show no significant correlation(P>0.05) with body weight of grass carp fed with two different level of sugar. But for the same genotype, the average body weight of group fed with normal sugar was heavier than that of group fed with high-level sugar. It was speculated that excess amount of sugar in feed can inhibit the growth of grass carp.