陕北白绒山羊碱性氨基酸转运载体基因cDNA克隆及时空表达分析

发布时间：2018-07-06 18:24

本文选题：陕北白绒山羊 + 碱性氨基酸转运载体　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：本研究旨在探讨陕北白绒山羊在吸收和转运碱性氨基酸的生理过程中,碱性氨基酸转运载体基因4F2hc(SLC3A2)、y+LAT1(SLC7A7)、y+LAT2(SLC7A6)、rBAT(SLC3A1)和CAT-1(SLC7A1)的主要表达部位和发育性表达规律,为进一步研究反刍动物氨基酸的跨膜转运机制,根据山羊的生理特点,科学配制饲粮提供理论支持。根据GenBank公布的绵羊4F2hc、y+LAT1、y+LAT2、rBAT和CAT-1基因cDNA序列设计引物,通过反转录PCR(Reverse Transcription PCR,RT-PCR)技术克隆得到上述基因cDNA全部或部分编码区序列(Coding Sequence,CDS),并进行序列分析;通过实时荧光定量PCR(Real-time Quantitative PCR,RT-QPCR)技术检测上述基因在3只1日龄陕北白绒山羊心脏、肝脏、肾脏、大脑、背最长肌、体侧皮肤、瘤胃、十二指肠、空肠、回肠和结肠共11各组织中mRNA的表达情况;屠宰健康1日龄、6月龄、8月龄、10月龄、12月龄陕北白绒山羊各3只,共15只,采集其十二指肠、空肠、回肠组织,探讨4F2hc、y+LAT1、y+LAT2、rBAT和CAT-1基因随山羊年龄增长在小肠不同肠段中的mRNA发育性表达规律。主要研究结果如下:1.成功克隆陕北白绒山羊4F2hc、y+LAT1、y+LAT2、rBAT基因cDNA全部CDS区序列及CAT1基因部分cDNA序列。4F2hc、y+LAT1、y+LAT2、rBAT和CAT-1基因系统进化上同绵羊、牛亲缘关系最近。2.4F2hc、y+LAT1、y+LAT2、rBAT和CAT-1基因在1日龄陕北白绒山羊各组织中mRNA的表达存在组织差异性。4F2hc mRNA在皮肤表达量显著高于其它组织(P0.05),除皮肤外,空肠表达量最高。y+LAT1 mRNA在小肠表达量显著高于其它组织(P0.05)。y+LAT2 mRNA在大脑和回肠中表达量显著高于其它组织(P0.05)。rBAT mRNA在肾脏表达量最高,小肠中也有较高表达。CAT-1 mRNA在大脑表达量最高,皮肤、回肠、肾脏也有相对较高的表达。3.4F2hc mRNA在陕北白绒山羊十二指肠、空肠和回肠表达量均以1日龄山羊为最高,其它月龄山羊相同肠段之间无显著差异(P0.05)。y+LAT1 mRNA在山羊十二指肠、空肠和回肠表达量随山羊年龄增加均呈逐渐降低的趋势。y+LAT2 mRNA在山羊十二指肠和空肠表达量随山羊年龄增加呈先升高后降低的趋势;回肠表达量以1日龄山羊为最低,其它各月龄山羊之间差异不显著(P0.05)。rBAT mRNA在十二指肠表达量以1日龄山羊为最高,其它各月龄山羊之间无显著差异(P0.05);空肠表达量随山羊年龄的增加均呈逐渐降低的趋势;回肠表达量在各年龄山羊之间无显著差异(P0.05)。CAT-1 mRNA在十二指肠和空肠表达量均以1日龄山羊为最低,回肠表达量以1日龄山羊为最高,其它各月龄山羊相同肠段之间无显著差异(P0.05)。综合以上试验结果,表明碱性氨基酸转运载体基因进化过程中在反刍动物间高度保守;各碱性氨基酸转运载体基因mRNA的主要表达部位存在差异,其在山羊小肠受肠段和发育阶段的影响,具有不同的发育性表达规律。
[Abstract]:The aim of this study was to investigate the main expression sites and developmental expression patterns of basic amino acid transport vector gene 4F2hc (SLC3A2), y LAT1 (SLC7A7), y LAT2 (SLC7A6), rBAT (SLC3A1) and CAT-1 (SLC7A1) during the physiological process of absorption and transport of basic amino acids in Northern Shaanxi white cashmere goats. In order to further study the transmembrane transport mechanism of amino acids in ruminants, theoretical support was provided according to the physiological characteristics of goats. Primers were designed according to the cDNA sequences of sheep 4F2hcCU y LAT1rBAT and CAT-1 gene published by GenBank. All or part of these cDNA sequences were cloned and sequenced by reverse transcription PCR (RT-PCR). Real-time quantitative PCR (Real-time quantitative PCR) was used to detect the above genes in the heart, liver, kidney, brain, longissimus dorsi, body skin, rumen, duodenum and jejunum of three 1-day-old Shaanxi white cashmere goats. The expression of mRNA in 11 tissues of ileum and colon was collected from duodenum, jejunum and ileum of 3 healthy white cashmere goats aged 10 months, 10 months and 12 months old, respectively, and the tissues of duodenum, jejunum and ileum were collected, and the tissues of duodenum, jejunum and ileum were collected. To investigate the developmental expression of 4F2hccnyLAT1rBAT and CAT-1 genes in different intestinal segments of the small intestine with the age of goats. The main results are as follows: 1. The complete CDS region and partial cDNA sequence of CAT1 gene were cloned successfully from 4F2hcCU y LAT1 + y LAT2rBAT gene of Northern Shaanxi White Cashmere Goat, and the phylogenetic characteristics of CAT1 gene and CAT-1 gene were the same as that of sheep. The expression of RBAT and CAT-1 genes in all tissues of Northern Shaanxi Cashmere Goat at 1 day of age was significantly higher than that in other tissues (P0.05), and the mRNA expression of 4F2hc mRNA in all tissues of Northern Shaanxi Cashmere Goat was significantly higher than that in other tissues (P 0.05), and the expression of CAT-1 gene in all tissues of Northern Shaanxi Cashmere Goat was significantly higher than that in other tissues (P0.05). The highest expression of YLAT1 mRNA in the jejunum was significantly higher in the small intestine than in other tissues (P0.05). The expression of yLAT2 mRNA in the brain and ileum was significantly higher than that in the other tissues (P0.05) .rBAT mRNA was the highest in the kidney, and the high expression of .CAT-1 mRNA in the small intestine was also the highest in the brain. The expression of 3.4F2hc mRNA in the duodenum, jejunum and ileum of the northern Shaanxi white cashmere goat was the highest in the 1-day-old goat, and there was a relatively high expression of 3.4F2hc mRNA in the skin, ileum and kidney. There was no significant difference between the same intestinal segments of other month old goats (P0.05). Y LAT1 mRNA was found in the duodenum of goats. The expression of yLAT2 mRNA in the duodenum and jejunum of goats increased at first and then decreased with the increase of goat age, and the expression level of ileum was the lowest in 1 day old goat. There was no significant difference in the expression of rBAT mRNA among other month old goats (P0.05). The expression of rBAT mRNA in the duodenum of 1-day-old goats was the highest (P0.05), but there was no significant difference among other month-old goats (P0.05), and the jejunum expression decreased gradually with the increase of goat age. The expression of CAT-1 mRNA in duodenum and jejunum was the lowest in 1-day-old goat, and the highest in 1-day-old goat (P0.05). The results showed that the basic amino acid transporter gene was highly conserved among ruminants during the evolution, and the main expression sites of the basic amino acid transporter gene mRNA were different among ruminants. It has different developmental expression patterns in goat small intestine affected by intestinal segment and developmental stage.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S827

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