CN110724747A - 一种与大黄鱼抗副溶血弧菌关联的snp位点、其筛选方法和应用 - Google Patents
一种与大黄鱼抗副溶血弧菌关联的snp位点、其筛选方法和应用 Download PDFInfo
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Abstract
本发明属于鱼类分子标记筛选技术领域,具体公开了一种与大黄鱼抗副溶血弧菌关联的SNP位点、其筛选方法和应用,SNP位点位于大黄鱼如SEQ ID NO:1所示的CTRP9基因cDNA序列上,且其1147位碱基处存在一个A或G的等位基因突变;经提取副溶血弧菌感染大黄鱼幼鱼的RNA并反转录成cDNA,设计如SEQ ID NO:2所述的正向引物和如SEQ ID NO:3所示的反向引物,以cDNA为模板进行PCR扩增反应,对目的条带清晰无杂带的PCR产物进行测序筛选得到。本发明中与大黄鱼抗副溶血弧菌相关的SNP位点可用于抗副溶血弧菌大黄鱼遗传育种,为大黄鱼的抗菌选育工作提供新思路。
Description
技术领域
本发明属于鱼类分子标记筛选技术领域,涉及一种与大黄鱼抗副溶血弧菌关联的单核苷酸多态性(SNP)分子标记,特别涉及一种位于大黄鱼CTRP9基因外显子上与抗副溶血弧菌关联的SNP位点、其筛选方法和应用。
背景技术
大黄鱼(Larimichthys crocea)属硬骨鱼纲、鲈形目、石首鱼科、黄鱼属,是我国近海主要养殖经济鱼类,近些年,由于其养殖环境恶化、养殖规模扩大、养殖密度过大等原因,导致其病害频发,尤其是副溶血弧菌(Vibrio parahaemolyticus)感染引起的弧菌病,对大黄鱼生长危害极大。弧菌病发病时间短、死亡快、传染性极强,严重威胁着渔业经济发展和人们的健康。因此,培育大黄鱼抗病品系是预防弧菌的重要途径之一。
单核苷酸多态性(single nucleotide polymorphism,SNP)是指在基因组水平上由单个核苷酸的变异所引起的DNA序列多态性,是一种理想的遗传标记。继限制性片段长度多态性(RFLP)、微卫星标记(SSR)后,SNP作为新一代分子标记,在遗传的多样性分析、关联分析、品种鉴定、高密度遗传连锁图谱的构建以及辅助育种等方面有广泛应用。目前尚未见到有关从免疫相关基因中筛选到与大黄鱼抗副溶血弧菌相关的SNP位点,以及应用的报道。
发明内容
本发明针对副溶血弧菌感染大黄鱼患弧菌病,提供一种位于大黄鱼CTRP9基因上与抗副溶血弧菌关联的SNP位点,确定大黄鱼免疫相关基因的SNP位点与抗副溶血弧菌关联,为大黄鱼抗副溶血弧菌的选育提供分子标记。
本发明中,与大黄鱼抗副溶血弧菌相关的SNP位点,位于大黄鱼如SEQ ID NO:1所示的CTRP9基因cDNA序列上,全长1368bp,编码455个氨基酸,其1147位碱基处存在一个A或G的等位基因突变,且该突变引起第383位氨基酸由异亮氨酸(Ile)突变为缬氨酸(Val)。
本发明还提供了上述与大黄鱼抗副溶血弧菌相关的SNP位点的筛选方法,包括以下步骤:
(ⅰ)用副溶血弧菌感染大黄鱼幼鱼后,提取其RNA并反转录成cDNA,其核苷酸序列如SEQ ID NO:1所示;
(ⅱ)设计引物对如SEQ ID NO:2所述的正向引物和如SEQ ID NO:3所示的反向引物,以cDNA为模板进行PCR扩增反应;
(ⅲ)经琼脂糖凝胶检测,对目的条带清晰无杂带的PCR产物进行测序,筛选得到上述SNP位点。
步骤(ⅱ)中,所述PCR扩增反应体系25uL为:2×Mix12.5uL,正向引物(10uM)和反向引物(10uM)各1uL,cDNA模板1uL,无菌水9.5uL;和/或
所述PCR扩增反应的程序依次为:95℃预变性5min,94℃变性30s、64℃退火30s、72℃延伸30s(45个循环),72℃终延伸10min。
本发明还提供了上述与大黄鱼抗副溶血弧菌相关的SNP位点在抗副溶血弧菌大黄鱼遗传育种上的用途。
与现有技术相比,本发明的有益效果是:
本发明对大黄鱼免疫相关基因非同义突变SNPs进行筛选与验证,SNPs标记不仅可以评估大黄鱼遗传多样性,而且为大黄鱼的抗病选育等工作提供研发基础。同时,本发明检测到大黄鱼CTRP9基因与副溶血弧菌抗性相关的SNP位点,为大黄鱼的抗菌选育工作提供新思路,有利于推进大黄鱼的遗传育种进程,提高大黄鱼养殖经济效益。
附图说明
图1是大黄鱼CTRP9基因的SNP位点峰型图,自上而下依次为副溶血弧菌易感组和抗副溶血弧菌组。
具体实施方式
下面结合附图和实施例对本发明进一步说明。
以下实例中大黄鱼CTRP9基因上与抗副溶血弧菌关联的SNP位点的筛选方法,通过用副溶血弧菌感染大黄鱼幼鱼,提取RNA并反转录成cDNA,采用引物设计、PCR扩增测序、SNP位点筛选获得SNP位点,利用SPSS18.0软件对感染前后抗感易感群体的基因型频率进行差异显著性分析,采用BioEdit软件进行分型获得与抗副溶血弧菌相关的SNP位点,具体步骤如下:
(1)大黄鱼暂养、驯化
出膜40天、1~2cm的大黄鱼苗由象山港水产引种育种有限公司赠送,放到1.0m(内径)×1.2m(高)的养殖桶里暂养3~4天,水体高1m,温度26℃,每天投饵2次(早上7:00、下午6:30),换水2次,每次换水90%,清理底部食物残渣及粪便。为了防止大黄鱼患白点病,换水时加入5ml甲醛,浓度为0.005‰。待鱼苗适应环境、进食稳定后,将暂养的大黄鱼移入实验缸中再进行实验。
(2)人工感染
实验水体为长80cm、宽45cm的玻璃缸,加入海水70L,每缸放苗500尾,水温控制在26℃,鱼苗稳定后,进行实验;设置对照组和副溶血弧菌(ATCC17802)感染实验组,浓度为1.5×107CFU/mL,实验组和对照组均设置3个平行组。各缸每隔24h投喂一次饵料,清理掉食物残渣和粪便,换水1/3,并补充感染菌以维持其恒定的浓度。每天清理、记录各缸中死苗数,RNA保护液保存死亡样本用于后续PCR验证。
(3)转录组分析
取实验第七天的对照组、副溶血弧菌感染患病的、副溶血弧菌感染未患病的样本各三条,Trizol法提取样本总RNA,混合后质检、建库,用TruSeq PE Cluster Kit在cBot中进行cluster generation,然后在Illumina Hiseq TM 2500中进行双向测序。测序得的rawreads去除低质量和错误碱基后得到clean reads,以模式生物斑马鱼转录组数据为参考序列进行比对,对测序数据的KEGG通路、差异表达基因和SNP进行分析。从转录组中筛选免疫相关的KEGG通路,根据通路中的免疫相关因子从差异表达基因中筛选差异的免疫基因,并筛选免疫基因的有义突变位点。
从抗副溶血弧菌组与对照组中筛选出127个免疫差异基因,其中有53个免疫基因上调,73个下调基因;53个免疫上调基因中共发现29个基因编码区存在146个SNP位点,其中非同义突变位点有28个。
(4)PCR扩增及测序
选择对照组、副溶血弧菌感染第四天死亡(易感)、第七天存活(抗感)的大黄鱼,各10尾、共30尾,分别提取整鱼的总RNA。以提取的RNA为模版用TaKaRa公司的PrimeScriptTMII1st and cDNA Synthesis Kit反转录成cDNA,-20℃保存备用。
设计引物对序列如下所示:
Primer F:AGCCTAACCTTCCTGTTCCTTTCTATACTATCATTT
Primer R:GGTGGTGTTGCCATCGGGACCTT。
采用上述引物以cDNA为模板进行PCR扩增,选择诺唯赞公司的2×Taq PlusMaster Mix(Dye Plus)试剂进行PCR反应,反应体系25uL:2×Mix12.5uL,Primer F(10uM)和Primer R(10uM)各1uL,cDNA模板1uL,无菌水9.5uL。扩增反应器为Eppendorf PCR仪(艾本德,德国)。PCR程序:95℃预变性5min,94℃变性30s、64℃退火30s、72℃延伸30s(45个循环),72℃终延伸10min。PCR产物经琼脂糖凝胶检测、目的条带清晰无杂带,剩余产物送上海生工生物工程股份有限公司进行测序。
(5)基因型的判定及数据分析
根据测序结果用BioEdit软件观察测序峰型图,进而确定SNPs位点的基因型;单峰为纯合子基因型,双峰为杂合子基因型。以上数据用SPSS18.0软件对对照组、第四天死亡样本(易感)、第七天存活样本(抗感)进行单因素方差分析(P<0.05:显著性差异;P<0.01:极显著差异)。结果发现:CTRP9基因型频率在副溶血弧菌抗感易感群体中存在极显著性差异,其他免疫基因如NF-kappa-B inhibitor zeta-like、glutathione S-transferase omega 1、galectin-4、tyrosine-protein kinase STYK1-like、APOBEC1 complementation factor、interferon reguLatory factor 2-binding protein 1-like、Arginase基因在副溶血弧菌实验组样本中不存在显著性差异。
(6)扩大样本检测CTRP9基因SNP位点
为了更准确验证CTRP9基因SNP位点在副溶血弧菌感染组样本中的情况,增加对照组样本至20尾、副溶血弧菌易感组至18尾、抗副溶血弧菌组至16尾,对CTRP9基因的两个SNP位点进一步验证。
如图1所示的副溶血弧菌易感组18个样本和抗副溶血弧菌组16个样本一代测序峰型图,SNP位点CTRP9-1147-A/G在副溶血弧菌易感组中的基因型频率分别为AA22.2%、AG77.8%、GG0,在抗副溶血弧菌组中AA37.5%、AG25%、GG37.5%。CTRP9基因的Ile突变为Val位点,副溶血弧菌易感组与抗副溶血弧菌组间存在极显著性差异,但是抗副溶血弧菌组与对照组不存在显著性差异,如表1所示。
表1:CTRP9基因在副溶血弧菌实验组SNP位点统计
由表1可知,SNP位点CTRP9-1147-A/G在副溶血弧菌抗感易感组基因型频率存在极显著性差异X2=11.879,P<0.01。表明SNP位点CTRP9-1147-A/G在副溶血弧菌抗感易感群体中存在极显著性差异,此位点与大黄鱼抗副溶血弧菌相关联。以上数据分析显示SNP位点CTRP9-1147-A/G在副溶血弧菌抗感和易感群体中基因型频率分别为GG37.5%和0,存在极显著性差异(P<0.01)。
SNP位点CTRP9-31147-GG基因型可以作为大黄鱼抗副溶血弧菌的候选基因,用于指导大黄鱼的分子标记辅助选择育种。与现有大黄鱼抗菌育种技术相比,从基因角度出发,本发明筛选到与大黄鱼抗副溶血弧菌相关的SNP位点,并把此位点运用在大黄鱼育种工作中,有望从遗传角度根本性的解决大黄鱼患弧菌病问题。
序列表
<110> 上海海洋大学
<120> 一种与大黄鱼抗副溶血弧菌关联的SNP位点、其筛选方法和应用
<141> 2019-11-18
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1368
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
atgctaaaca acactgcccc agggactggc tttggtgtgg acaacaccat tgattggaga 60
ggctcgggct cgaactcggg ctcggactcg ggctcggact cggactcgga ctcagatgaa 120
agtggggggc acttcccctt taatgcacgt gcccgtcatc accacatgcc ggggccaccc 180
agcaacatga cccggatgat gatgagagaa gatgacatgc ccattttacc tgatctatca 240
atctgtgaca tgctgttgag ctcacctaac ccaccaccta tcaacgagat tccttttttc 300
tgcctctgtt cccactgtaa gggtaccgtg ggacccaaag gagaccgcgg agaccgaggt 360
ctcccaggtc agaagggcga cgttggggaa caggggatgc ctggatctat gggtttcact 420
gggatgaagg gcgagcgagg cttcaaagga gaaaaaggtg acagaggaac ggcaggcccc 480
caaggtcaac aaggccccca gggagaaacc ggcatatgcc ctgcttcctg cgatagtgtc 540
cagggtccac caggtgaaca aggcccctct gggccagccg gttctcgggg cctgcctgga 600
atcaagggag aaaggggacc catgggtttt aagggtgata aaggtgacct gggcatacct 660
ggcaaccctg gggtggacgg ccagaagggt gatcaaggta tgcaggggat atgtgagtgc 720
acagatgggg aagacggcgc taatggggca gcaggagcaa agggggataa aggggataaa 780
ggtgacactg gcccccgggg tgcacagggt actatggggc taaaaggcaa catgggtgcc 840
atgggtatga tggggcagcc tgggccgtgc tccccaacca tcaaatcagc attctccgcg 900
gcaatcaacc agtcgtttcc agagcctaac cttcctgttc ctttctatac tatcatttat 960
aataaggaaa accatttcga tccaagaggc atgtacacag cccctgttaa tggcacatac 1020
gtcttcacct tcaacatggc agttgctgaa aggactctta aggttggcct gttccaaaac 1080
ttctggccta taatcaaagc aacggaagtg gtaggaaacg agcagtcatc cgtgagccag 1140
actatcatcc tccacctcaa aaggtttgac cgggtgtggc tgcaggtgaa gagcagcacc 1200
accaacggca tgtacaccga cagtgagagc agcagcacgt tctctgggtg gctgcagttc 1260
cctgacagct gcgatccgcc cctgggcaga cagatgtttc ctccacaaga tgaccgagac 1320
tttagctggg aaggtcccga tggcaacacc acccctccac catactag 1368
<210> 2
<211> 36
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
agcctaacct tcctgttcct ttctatacta tcattt 36
<210> 3
<211> 23
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 3
ggtggtgttg ccatcgggac ctt 23
Claims (5)
1.一种与大黄鱼抗副溶血弧菌相关的单核苷酸多态性(SNP)位点,其特征在于,所述SNP位点位于大黄鱼如SEQ ID NO:1所示的CTRP9基因cDNA序列上,且其1147位碱基处存在一个A或G的等位基因突变。
2.权利要求1所述SNP位点的筛选方法,其特征在于,包括以下步骤:
(ⅰ)用副溶血弧菌感染大黄鱼幼鱼后,提取其RNA并反转录成cDNA,其核苷酸序列如SEQID NO:1所示;
(ⅱ)设计如SEQ ID NO:2所述的正向引物和如SEQ ID NO:3所示的反向引物的引物对,以步骤(ⅰ)中cDNA为模板进行PCR扩增反应;
(ⅲ)经琼脂糖凝胶检测,对步骤(ⅱ)中目的条带清晰无杂带的PCR产物进行测序,筛选得到所述SNP位点。
3.根据权利要求2所述SNP位点的筛选方法,其特征在于,步骤(ⅱ)中,所述PCR扩增反应体系为:2×Mix12.5uL、浓度均为10uM的所述正向引物和反向引物各1uL、所述cDNA模板1uL和无菌水9.5uL。
4.根据权利要求2所述SNP位点的筛选方法,其特征在于,步骤(ⅱ)中,所述PCR扩增反应的程序依次为:95℃预变性5min,94℃变性30s、64℃退火30s、72℃延伸30s(45个循环),72℃终延伸10min。
5.权利要求1所述与大黄鱼抗副溶血弧菌相关的SNP位点在抗副溶血弧菌大黄鱼遗传育种上的用途。
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20150099695A1 (en) * | 2012-05-25 | 2015-04-09 | The Johns Hopkins University | C1q/TNF-RELATED PROTEIN-9 (CTRP9) AND USE IN PREVENTION AND TREATMENT OF METABOLIC DISORDERS |
CN110205389A (zh) * | 2019-05-31 | 2019-09-06 | 宁波大学 | 缢蛏抗副溶血弧菌相关SNPs分子标记及其扩增引物和应用 |
-
2019
- 2019-11-18 CN CN201911128193.8A patent/CN110724747A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20150099695A1 (en) * | 2012-05-25 | 2015-04-09 | The Johns Hopkins University | C1q/TNF-RELATED PROTEIN-9 (CTRP9) AND USE IN PREVENTION AND TREATMENT OF METABOLIC DISORDERS |
CN110205389A (zh) * | 2019-05-31 | 2019-09-06 | 宁波大学 | 缢蛏抗副溶血弧菌相关SNPs分子标记及其扩增引物和应用 |
Non-Patent Citations (5)
Title |
---|
EUKARYOTA等,: ""PREDICTED:Larimichthys crocea complement C1q tumor necrosis factor-related protein 2-like(LOC104937181),mRNA"", 《GENBANK》 * |
孙明洁等: ""两种弧菌感染大黄鱼免疫相关基因的SNP位点分析"", 《上海海洋大学学报》 * |
池信才等: "大黄鱼病原副溶血弧菌单克隆抗体制备及其应用", 《海洋科学》 * |
王国良等: "养殖大黄鱼(Pseudosciaena crocea)3种致病弧菌的分子鉴定及其系统发育学分析", 《海洋与湖沼》 * |
赵现锋等: "水产品中副溶血性弧菌快速检测技术研究进展", 《食品安全质量检测学报》 * |
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