草鱼肝脏型醛缩酶基因突变影响糖代谢分子机制及应用

The glucose are the least expensive form of dietary energy in the natural world. High-glucose feeds can affect growth, feed utilization and so on. The grass carp was selected as a model owing to its relatively high glucose-metabolizing capability. We had previously obtained a haplotype with one exon missense mutation (C+1042A) and intron mutation (A117C) completely linked of aldolase B in grass carp, and the AA/AA genotype individuals showed significant growth advantage. Aldolase is a key enzyme in glycolysis and gluconeogenesis. It is speculated that mutations affect enzyme activity, affect the glucose metabolism pathway, and indirectly affect growth. In this study, C+1042A genotypes of CC and AA were obtained as eukaryotic expression products, and the effects of mutations on the enzyme activity were determined; then parents of heterozygous pedigrees were constructed to analyze the differences in gene expression and mutations of offspring on the glucose metabolism pathway and growth axis gene expression. The influence of quantity on the molecular mechanism of the effect of gene mutation on glucose utilization. The glucose level- genotype- growth trait- gene expression correlation was analyzed by a mixed linear model to assess the glucose tolerance potential of different haplotypes. This study is of great significance in improving the utilization rate of grass carp glucose and reducing the cost of feed. It also provides a theoretical basis for the breeding of resistant glucose of grass carp.

糖类是自然界丰富而廉价的饲料能量来源，高糖饲料易造成鱼类生长慢，抗逆性差等。草鱼是典型的植食性鱼类，是耐糖遗传解析重要模式鱼类。申请人前期在草鱼肝脏型醛缩酶（ALDB）上获得1个外显子错义突变（C+1042A）和内含子突变（A117C）完全连锁的单倍型，AA/AA基因型个体生长优势显著。醛缩酶是糖酵解和糖异生途径正负反馈调节关键酶，推测基因突变影响酶活，对糖代谢通路有影响，间接影响了生长。本项目分别获得C+1042A基因型为CC和AA真核表达产物，检测突变对酶活力影响；然后构建亲本杂合型家系，分析后代突变型基因表达差异和突变对糖代谢通路和生长轴基因表达量影响，从分子水平上解析基因突变对糖利用影响的分子机制。并通过混合线性模型分析糖水平-基因型-生长性状-基因表达相关性，评估不同单倍型的耐糖潜力。本研究在提高草鱼糖利用率、降低饲料成本等方面具有重要意义，也为耐糖草鱼新品种选育奠定理论依据。

高糖饲料易造成鱼类生长慢，抗逆性差等，草鱼是典型的植食性鱼类，是耐糖遗传解析重要模式鱼类。草鱼肝脏型醛缩酶（ALDB）存在错义突变（C+1042A）和内含子突变（A117C）完全连锁的单倍型，单倍型分别为AAAA、ACAC和CCCC基因型，因其完全连锁，将单倍型简称为AA、AC和CC基因型，其中CC基因型个体生长优势显著。为了解析醛缩酶CC基因型影响生长的机制。项目采用真核表达载体获得两种突变蛋白，酶活测定表明AA突变型和CC突变型酶浓度为2.82ng/ml和4.72ng/ml；构建杂合型亲本自交家系，采用高糖和普通糖进行3个月养殖，对AA、AC和CC基因型对应的个体的ALDB、FDB、PKRL、GH、GHR、IGF-1、SS1、SS2、SS3、Myostatin、Myod基因进行qPCR分析。表明ALDB基因表达量与GHR基因显著正相关（0.01<P<0.05），与SS-1基因显著负相关（0.01<P<0.05）；构建杂合型亲本自交家系，用普通糖、中高糖和高糖饲料进行3个月养殖。经分析普通糖水平CC基因型个体分别比AC和AA基因型个体增重快3.83%和6.63%；中高糖水平CC基因型个体分别比AC和AA基因型个体增重快6.90%和20.65%；高糖水平CC基因型个体分别比AC和AA基因型个体增重快6.29%和14.5%。表明ALDB基因CC基因型在高糖水平比其它基因型增重快6.29%以上，但仍旧低于普通糖水平AA基因型增重水平。且高糖水平GH水平显著低于普通糖水平，表明高糖还是抑制生长速度。项目从基因和蛋白水平评估高糖和普通糖水平草鱼生长相关基因表达量，和分析了ALDB基因CC基因型生长速度具有优势的原因。且证明该基因型个体生长速度和耐糖能力均较强，可以通过选择仅含有CC基因型的个体做亲本，其后代耐糖潜力至少为6.29%以上，该结果为耐糖草鱼新品种选育奠定理论依据。