CN112480229B - 蛋白Secp43或其编码基因在调控黑腹果蝇雄性生育中的应用 - Google Patents
蛋白Secp43或其编码基因在调控黑腹果蝇雄性生育中的应用 Download PDFInfo
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- CN112480229B CN112480229B CN202011353476.5A CN202011353476A CN112480229B CN 112480229 B CN112480229 B CN 112480229B CN 202011353476 A CN202011353476 A CN 202011353476A CN 112480229 B CN112480229 B CN 112480229B
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Abstract
本发明公开了蛋白Secp43或其编码基因在调控黑腹果蝇雄性生育中的应用。所述的蛋白Secp43,其氨基酸序列如SEQ ID NO.2所示。本发明通过发现,敲除编码基因Secp43可以使得黑腹果蝇雄性不育,其成束精子无法完成分离,未成熟的精子则无法进入储精囊,这可能是雄性不育的主要原因。因此本发明筛选得到一个黑腹果蝇雄性不育相关的新蛋白,鉴于果蝇和人类部分蛋白的同源性,将有助于我们寻找到新的不孕不育的治疗靶点。并且从RNA结合蛋白角度对Secp43造成黑腹果蝇雄性不育的调控机理进行深入研究,将有助于我们寻找到新的不孕不育的治疗靶标。
Description
技术领域:
本发明属于生物技术领域,具体地涉及蛋白Secp43或其编码基因在调控黑腹果蝇雄性生育中的应用。
背景技术:
在真核生物中,基因表达的转录后调节是一个非常复杂和关键的步骤,在RNA代谢途径中,都需要大量蛋白质因子相互作用,同时RNA代谢还受到多种信号途径的调节。根据RNA的功能和位置可以把RNA分为很多种类,每一种RNA都有大量RNA结合蛋白和它相互作用来稳定、保护、组装或者转移它,同时还调节RNA同其它分子的相互作用或者催化RNA解螺旋和复制等。RNA结合蛋白的研究,历来受到相关生物化学家们的重视,已经有很多不同结构和功能的RNA结合蛋白已经被鉴定出来。目前研究得比较清楚的RNA结合蛋白有核糖核蛋白(RNP)、KH基序RNA结合蛋白、双螺旋RNA结合蛋白和冷激基序RNA结合蛋白(Graumann andMarahiel,1998)。然而,还有大量RNA结合蛋白的功能尚未清楚。
近年来,对动植物中RNA结合蛋白的研究有很多,但对RNA结合蛋白的生理功能和分子功能研究还有待深入。RNA结合蛋白和动物的神经系统发育密切相关。目前,RNA结合蛋白在神经退行性疾病的发生中所起到的作用受到广泛关注。在2006年和2009年,TDP-43和FUS相继被报道与渐冻人症和其他几种神经退行性疾病的发生密切相关(Kwiatkowski etal.,2009;Neumann et al.,2006)。TDP-43和FUS都是RNA结合蛋白,具有RRM结构域和甘氨酸富集结构域(glycine rich domain,GRD),均属于hnRNP家族。TDP-43和FUS作为RNA结合蛋白在RNA转录后调控上发挥着重要作用,大量证据表明它们的功能异常可能与神经退行性疾病的发生紧密相关。
此外,在肿瘤发生和调节机制上对RNA结合蛋白的研究也很多。Charles等对CRD-BP进行研究发现,CRD-BP是一种多功能的RNA结合蛋白,它结合c-myc、肌动蛋白mRNAs、胰岛素样生长因子结合蛋白ⅡmRNAs和H19 RNA(Liao et al.,2004)。CRD-BP在胎儿中含量很高但在成人中很低或者没有,他们在一些成年人的乳房、结肠和肺部中检测到CRD-BP的表达,且这这些部位产生癌变的概率很高。因此他们推测CRD-BP涉及肿瘤的形成。
由于生殖发育一直是人类医学关注的重点之一,其不孕不育的治疗靶点应该多种多样。
发明内容:
本发明的第一个目的是提供蛋白Secp43或其编码基因Secp43在调控黑腹果蝇雄性生育中的应用,所述的蛋白Secp43,其氨基酸序列如SEQ ID NO.2所示。
优选,所述的蛋白Secp43的编码基因Secp43,其核苷酸序列如SEQ ID NO.1所示。
优选,敲除编码基因Secp43在调控黑腹果蝇雄性不育中的应用。
所述的敲除编码基因Secp43优选是利用Crispr/Cas9特异性敲除编码基因Secp43,其中用于靶向编码基因Secp43的gRNA,其核苷酸序列如SEQ ID NO.3所示。
本发明的第二个目的是提供一种在利用Crispr/Cas9特异性敲除编码基因Secp43中用于靶向编码基因Secp43的gRNA,其核苷酸序列如SEQ ID NO.3所示。
本发明的第三个目的是提供上述gRNA在制备敲除编码基因Secp43从而调控黑腹果蝇雄性不育的制剂中的应用。
本发明通过发现,敲除编码基因Secp43可以使得黑腹果蝇雄性不育,其成束精子无法完成分离,未成熟的精子则无法进入储精囊,这可能是雄性不育的主要原因。因此本发明筛选得到一个黑腹果蝇雄性不育相关的新蛋白,鉴于果蝇和人类部分蛋白的同源性,将有助于我们寻找到新的不孕不育的治疗靶点。并且从RNA结合蛋白角度对Secp43造成黑腹果蝇雄性不育的调控机理进行深入研究,将有助于我们寻找到新的不孕不育的治疗靶标。
附图说明:
图1是Secp43的杂合突变体检测图。箭头表示突变体。NC表示野生型果蝇经过PCR扩增与酶切之后的结果;
图2是黑腹果蝇的生育力统计。每个点表示平均数±SEM(n=30),字母表示多重分析结果(P<0.05,统计方法为LSR,SPSS);
图3是敲除Secp43后黑腹果蝇雄性成虫精巢的解剖图。
具体实施方式:
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
实施例1
1.目标基因,其核苷酸序列如下所示:
atggcgtccgtgcattgtcaactgtggatgggaagcctggagtcctacatgacggagaacttcataatcgccgctttccggaagatgggcgaagatcccaccacggtgcgcctgatgcgcaacaagtacacgggcgaaccggccggctactgcttcgtcaacttcatatccgatgaccatgcgctggacgcgatgcacaagctaaacggtaagcccattccgggcaccaatcccattgtgcgattccgtcttaactcggccagcaactcgtacaagctgcccgggaatgaacgcgaatttagcgtctgggtgggcgacctcagctcggacgtggacgactatcagctgtacaaggtgttctccagcaagttcacatcgatcaaaaccgccaaggtgatcttggacagcctggggttttccaagggttacggctttgtgcgatttggcatcgaggatgagcagaaatcagcgctgtacgacatgaacgggtacatcggattgggtaccaagcccataaagatttgcaatgccgtgcccaaacccaaatccgagttgggtggtgctgtgggcgaaggcaacacaaactatggatatggaagcggtatgactgcagcaggtggcaccgactactcgcagtactacgaccccaccagcacctattggcagggataccaggcctggcaaggttactacgagcaggccggtgcttcgatcacagatgcggctgcatactaccagcaggccatgtcgcagtcgcactctaatccgcagactctggcccagcacgccgaggcctggagcgcacaacgcagcgcccagtacgagcagcagcagcagcagcagaccgcttccgccgccaatggagctgaggatgagaacggactggtggagcacaagttcgtcctggatgtggacaaactgaaccgagaggccattgatgccgaccgccgtctgtacgatgctctggagagctctaagtggctgcccattgagcagctggaagtcttttag(具体序列如SEQ ID NO.1所示),其编码的蛋白的氨基酸序列如SEQ ID NO.2所示,命名为蛋白Secp43。
登陆Crispr在线gRNA靶标网站选取合适的gRNA,http:// tools.flycrispr.molbio.wisc.edu/targetFinder/#userconsent#,蛋白通常只需1个gRNA即可产生失效蛋白。设计时,在gRNA的两端各加上了bbs I酶切后的序列。此外,为了便于快速检测突变体,我们选择target部位含有常用酶切位点gRNA序列,如发生突变,酶切则失效,反之则反。我们为Secp43选择了1个gRNA(靶点),序列如下:
gRNA:5’-ATGGGCGAAGATCCCACCA-3’,其核苷酸序列如SEQ ID NO.3所示。
2.gRNA单体质粒的构建:包括短引物替换法构建gRNA单体、pMD18T-U6-Bbs I载体酶切、载体与gRNA片段的连接、转化及重组质粒的筛选鉴定。具体如下:
(1)短引物替换法构建gRNA单体。
1)将合成的引物(FP:GTCGATGGGCGAAGATCCCACCA;RP:AAACTGGTGGGATCTTCGCCCAT)成对摆放好,13000g离心2min。
2)加入TE Buffer将引物稀释至100μM。稀释完毕后各取2μl混合,成为终浓度为10μM的引物。
3)94℃高温处理5min使引物复性结合,冰上备用。
(2)酶切载体(pMD18T-U6-Bbs I)
将反应物混匀后稍离心,37℃水浴中酶切2-4h。
(3)载体与gRNA片段的连接、转化及重组质粒的筛选鉴定
将gRNA目的片段和pMD18-T-U6载体利用T4 DNA连接酶进行体外连接,反应体系如下:
将反应物混匀后,稍离心,16℃连接2h。然后将连接产物转化进表达菌大肠杆菌DH5a中,通过PCR鉴定gRNA是否插入载体,并进行测序验证,将鉴定正确的菌液制成甘油菌,-80℃冰箱保存。
所述的pMD18-T-U6载体是在pMD18-T中的bbs I酶切位插入以下序列:
5'-GCTCACCTGTGATTGCTCCTACTCAAATACAAAAACATCAAATTTTCTGTCAATAAAGCATATTTATTTATATTTATTTTACAGGAAAGAATTCCTTTTAAAGTGTATTTTAACCTATAATGAAAAACGATTAAAAAAAATACATAAAATAATTCGAAAATTTTTGAATAGCCCAGGTTGATAAAAATTCATTTCATACGTTTTATAACTTATGCCCCTAAGTATTTTTTGACCATAGTGTTTCAATTCTACATTAATTTTACAGAGTAGAATGAAACGCCACCTACTCAGCCAAGAGGCGAAAAGGTTAGCTCGCCAAGCAGAGAGGGCGCCAGTGCTCACTACTTTTTATAATTCTCAACTTCTTTTTCCAGACTCAGTTCGTATATATAGACCTATTTTCAATTTAACGTCGGGGTCTTCGAGAAGACCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTGCCTACCTGGAGCCTGAGAGTTGTTCAATAAAATAAAAATGTTTCGTTTTTTTGCTTTCGCCAGTATTTATTATTTTTCATCAATATGTATTCAATTTGGTATGTATTTAGTAATTGTAATATATAGACAATGGTTTTCCGTTGACG-3'
3.PUAST-attB注射载体的构建:包括将单体质粒(步骤2最后构建的保存在甘油菌中的含gRNA片段的重组质粒)亚克隆到Gateway载体中和转化及重组质粒的筛选鉴定。具体如下:
(1)将单体质粒亚克隆到Gateway载体中
将反应物混匀后稍离心,室温反应1-2h。
(2)转化及重组质粒的筛选鉴定
将反应产物加入1ul蛋白酶K于37℃水浴10min,转化进表达菌大肠杆菌DH5a中,通过EcoR I和BamH I酶切鉴定3个gRNA是否插入载体,将鉴定正确的菌液制成甘油菌,-80℃冰箱保存,提取其中重组质粒可用于注射。
4.突变体果蝇的制备及杂交。
(1)注射前胚胎的收集和处理
①在大培养瓶中收集300只左右的待注射果蝇,保证雌果蝇与雄果蝇比例在2:1,果蝇年龄控制在3天以内,让其交配充分。
②交配2天之后,开始收集胚胎。收集时将果蝇倒入收卵器中,收集卵的果汁平皿涂上一层糊状的酵母。将瓶子倒置放在安静的环境中,25℃静置。
③每隔1-2h即可更新果汁平皿,将收集到的卵在显微镜下剥壳,按统一方向排列整齐后,粘到带有双面胶的盖玻片上,在硅胶中干燥2-3分钟,以减少卵的内压。干燥以后,用一薄层的700卤化碳油盖好。
(2)注射针头的制备
用拉针仪将显微注射用的玻璃毛细针管加热拉制针头。在针头中灌入待注射溶液,将针头在显微镜盖玻片的周围轻蹭一下,以此折断针头保证液体流出。
(3)显微注射操作
显微注射在18℃(以减缓发育的速度)、相对湿度大于70%的房间内完成。将带有胚胎的载玻片置于倒置显微镜下,注射的针头入针点可定在胚胎长的5%-10%处,可以移动显微操作载物台来调节位置。在胚胎的尾部前三分之一进行注射(注射的是上述含有gRNA的重组质粒,浓度500ng/ul,体积大约1-2nl)。注射结束后,将带有胚胎的盖玻片放到湿润的培养皿中,置于18摄氏度的培养箱中使其继续发育。
(4)转基因果蝇的饲养和筛选
注射后24-36小时,胚胎即可发育为1龄幼虫,将这些幼虫用毛刷轻挑至带有酵母的玉米培养基中,约50只幼虫一瓶。十天以后,待果蝇羽化后尽快挑出避免其互相交配,并使每一只羽化果蝇与野生型w1118果蝇杂交,w1118的雌果蝇应挑选处女蝇。约两周后,检查杂交果蝇的F1代,从中挑选眼色为橘黄色的果蝇,可初步认定为转基因果蝇。
(5)将转基因鉴定成功的带有橘黄色眼标记的gRNA果蝇与nos-Cas9或vasa-Cas9果蝇(能表达CAS9)杂交,方案如下(以lncRNA在2号染色体,注射86F为例),获得非致死纯合子转基因果蝇:
5.突变体果蝇的PCR检测及测序
提取非致死纯合子转基因果蝇的DNA,设计引物,使得每对引物包含gRNA靶标位点,通过PCR扩增出目的条带,然后利用设计初时选择的限制性内切酶对上述片段进行酶切反应,检测2条染色体的序列缺失情况。如果有一条染色体发生序列突变,酶切则失败,反之则反。Secp43的扩增引物如下:
F:TACAGCTGATAGTCGTCCAC
Secp43(CG15440)
R:GACACCAGGTCAGCATTTGA
实施例2
(1)设计引物,使得每对引物包含gRNA靶标位点,通过PCR扩增该片段。由于突变位置具有酶切位点,因此仅有一条染色体的PCR片段会被顺利酶切,另外一条发生突变的染色体的PCR产物则不会被酶切。结果表明,我们获得了Secp43的杂合突变体,如图1所示。结果表明,突变体产生率约为10%,最后通过测序确定缺失位置和缺失碱基数。
(2)随机挑选羽化相同天数的野生型黑腹果蝇w1118、Secp43的移码突变体Secp43L2-6和Secp43L5-4、Secp43的非移码突变体Secp43L3-3和Secp43L10-2各30只刚羽化的成虫(雌成虫和雄成虫),分别单头与3只w1118进行交配,3D后统计其生育能力。实验结果显示(图2),Secp43的移码突变体雄性成虫后代数为零,而移码突变体雌性成虫的后代数与野生型之间并无显著差异。此外,Secp43的非移码突变体Secp43L3-3和Secp43L10-2的雄成虫与雌成虫的后代数与野生型相比则无明显变化,表明Secp43对雄性黑腹果蝇的生育能力起关键作用。
(3)挑选充分交配的野生型黑腹果蝇w1118、Secp43的移码突变体Secp43L2-6,解剖其精巢,观察精子的形成和发育状况,详见图3。我们发现,正常野生型果蝇的精巢内精子的分布均匀且充盈,16个初级精母细胞经过两轮减数分裂形成64个成束精子并分离,分离后的成熟精子可以进入储精囊,然而,Secp43的移码突变体中,成束精子无法完成分离,未成熟的精子则无法进入储精囊,我们推测这是雄性不育的主要原因。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均因为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110> 广东省农业科学院植物保护研究所
<120> 蛋白Secp43或其编码基因在调控黑腹果蝇雄性生育中的应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1011
<212> DNA
<213> 黑腹果蝇(Drosophila melanogaster)
<400> 1
atggcgtccg tgcattgtca actgtggatg ggaagcctgg agtcctacat gacggagaac 60
ttcataatcg ccgctttccg gaagatgggc gaagatccca ccacggtgcg cctgatgcgc 120
aacaagtaca cgggcgaacc ggccggctac tgcttcgtca acttcatatc cgatgaccat 180
gcgctggacg cgatgcacaa gctaaacggt aagcccattc cgggcaccaa tcccattgtg 240
cgattccgtc ttaactcggc cagcaactcg tacaagctgc ccgggaatga acgcgaattt 300
agcgtctggg tgggcgacct cagctcggac gtggacgact atcagctgta caaggtgttc 360
tccagcaagt tcacatcgat caaaaccgcc aaggtgatct tggacagcct ggggttttcc 420
aagggttacg gctttgtgcg atttggcatc gaggatgagc agaaatcagc gctgtacgac 480
atgaacgggt acatcggatt gggtaccaag cccataaaga tttgcaatgc cgtgcccaaa 540
cccaaatccg agttgggtgg tgctgtgggc gaaggcaaca caaactatgg atatggaagc 600
ggtatgactg cagcaggtgg caccgactac tcgcagtact acgaccccac cagcacctat 660
tggcagggat accaggcctg gcaaggttac tacgagcagg ccggtgcttc gatcacagat 720
gcggctgcat actaccagca ggccatgtcg cagtcgcact ctaatccgca gactctggcc 780
cagcacgccg aggcctggag cgcacaacgc agcgcccagt acgagcagca gcagcagcag 840
cagaccgctt ccgccgccaa tggagctgag gatgagaacg gactggtgga gcacaagttc 900
gtcctggatg tggacaaact gaaccgagag gccattgatg ccgaccgccg tctgtacgat 960
gctctggaga gctctaagtg gctgcccatt gagcagctgg aagtctttta g 1011
<210> 2
<211> 336
<212> PRT
<213> 黑腹果蝇(Drosophila melanogaster)
<400> 2
Met Ala Ser Val His Cys Gln Leu Trp Met Gly Ser Leu Glu Ser Tyr
1 5 10 15
Met Thr Glu Asn Phe Ile Ile Ala Ala Phe Arg Lys Met Gly Glu Asp
20 25 30
Pro Thr Thr Val Arg Leu Met Arg Asn Lys Tyr Thr Gly Glu Pro Ala
35 40 45
Gly Tyr Cys Phe Val Asn Phe Ile Ser Asp Asp His Ala Leu Asp Ala
50 55 60
Met His Lys Leu Asn Gly Lys Pro Ile Pro Gly Thr Asn Pro Ile Val
65 70 75 80
Arg Phe Arg Leu Asn Ser Ala Ser Asn Ser Tyr Lys Leu Pro Gly Asn
85 90 95
Glu Arg Glu Phe Ser Val Trp Val Gly Asp Leu Ser Ser Asp Val Asp
100 105 110
Asp Tyr Gln Leu Tyr Lys Val Phe Ser Ser Lys Phe Thr Ser Ile Lys
115 120 125
Thr Ala Lys Val Ile Leu Asp Ser Leu Gly Phe Ser Lys Gly Tyr Gly
130 135 140
Phe Val Arg Phe Gly Ile Glu Asp Glu Gln Lys Ser Ala Leu Tyr Asp
145 150 155 160
Met Asn Gly Tyr Ile Gly Leu Gly Thr Lys Pro Ile Lys Ile Cys Asn
165 170 175
Ala Val Pro Lys Pro Lys Ser Glu Leu Gly Gly Ala Val Gly Glu Gly
180 185 190
Asn Thr Asn Tyr Gly Tyr Gly Ser Gly Met Thr Ala Ala Gly Gly Thr
195 200 205
Asp Tyr Ser Gln Tyr Tyr Asp Pro Thr Ser Thr Tyr Trp Gln Gly Tyr
210 215 220
Gln Ala Trp Gln Gly Tyr Tyr Glu Gln Ala Gly Ala Ser Ile Thr Asp
225 230 235 240
Ala Ala Ala Tyr Tyr Gln Gln Ala Met Ser Gln Ser His Ser Asn Pro
245 250 255
Gln Thr Leu Ala Gln His Ala Glu Ala Trp Ser Ala Gln Arg Ser Ala
260 265 270
Gln Tyr Glu Gln Gln Gln Gln Gln Gln Thr Ala Ser Ala Ala Asn Gly
275 280 285
Ala Glu Asp Glu Asn Gly Leu Val Glu His Lys Phe Val Leu Asp Val
290 295 300
Asp Lys Leu Asn Arg Glu Ala Ile Asp Ala Asp Arg Arg Leu Tyr Asp
305 310 315 320
Ala Leu Glu Ser Ser Lys Trp Leu Pro Ile Glu Gln Leu Glu Val Phe
325 330 335
<210> 3
<211> 19
<212> DNA
<213> 黑腹果蝇(Drosophila melanogaster)
<400> 3
atgggcgaag atcccacca 19
Claims (5)
1.蛋白Secp43或其编码基因Secp43在调控黑腹果蝇雄性生育中的应用,所述的蛋白Secp43,其氨基酸序列如SEQ ID NO.2所示。
2.根据权利要求1所述的应用,其特征在于,所述的蛋白Secp43的编码基因Secp43,其核苷酸序列如SEQ ID NO.1所示。
3.敲除编码基因Secp43在调控黑腹果蝇雄性不育中的应用,所述的编码基因Secp43,其核苷酸序列如SEQ ID NO.1所示。
4.根据权利要求3所述的应用,其特征在于,所述的敲除编码基因Secp43是利用Crispr/Cas9特异性敲除编码基因Secp43,其中用于靶向编码基因Secp43的gRNA,其核苷酸序列如SEQ ID NO.3所示。
5.gRNA在制备敲除编码基因Secp43从而调控黑腹果蝇雄性不育的制剂中的应用,所述的gRNA,其核苷酸序列如SEQ ID NO.3所示。
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