CN113481207B - 家蚕卵滞育的必需基因Bmtret1及其应用 - Google Patents
家蚕卵滞育的必需基因Bmtret1及其应用 Download PDFInfo
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- CN113481207B CN113481207B CN202110801492.4A CN202110801492A CN113481207B CN 113481207 B CN113481207 B CN 113481207B CN 202110801492 A CN202110801492 A CN 202110801492A CN 113481207 B CN113481207 B CN 113481207B
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Abstract
本发明涉及一个家蚕卵滞育的必需基因Bmtret1及其应用,属于家蚕技术研究领域。本发明通过靶向家蚕Bmtret1基因,降低其表达量,或者破坏Bmtret1基因结构,可以使本应进入滞育的蚕卵转变为非滞育卵。这一发明可以用于在蚕业生产中培育滞育性可调节的家蚕材料和品种,同时为通过调控鳞翅目昆虫的卵滞育实现害虫防治提供一种新的思路。
Description
技术领域
本发明属于家蚕技术研究领域,具体涉及一个家蚕卵滞育的必需基因Bmtret1及其应用。
背景技术
昆虫在自然选择的过程中,形成了多种适应周期性不利环境的生存策略。滞育是昆虫在某个特定的生长阶段,主动停止发育以度过不良环境的一种季节性策略。对昆虫滞育的遗传基础进行研究,不仅有助于理解导致昆虫滞育的原因,为昆虫滞育研究提供新的思路,还能为鳞翅目害虫防控提供潜在的分子靶标,具有重要的科学意义及应用价值。
根据滞育发生的时期,可分为胚胎滞育、幼虫滞育、蛹滞育和成虫滞育。家蚕是典型的胚胎滞育昆虫,具有多种一化性、二化性、及多化性品系,能够为滞育的研究提供丰富的材料,是研究胚胎滞育的重要模式生物。
家蚕的胚胎滞育由母性效应和胚胎自体基因型共同控制,目前对家蚕滞育的研究主要集中于母代环境诱导和化性相关基因造成咽下神经节分泌DH量不同从而影响子代滞育的母性效应方面,以及滞育卵与非滞育卵中脂类和糖类物质代谢等方面。而对子代胚胎自体基因型即受精后的滞育决定因子是什么,如何调控滞育的研究较少。
在家蚕基因功能研究中,通常需要通过胚胎注射来实现转基因、基因干涉和基因敲除。如材料为滞育卵,在注射后需用高氧、电激或调节母代催青温度(二化性)等方法解除滞育,会对蚕卵造成伤害或出现解除滞育不完全的情况;如果材料为多化性非滞育卵,则需要对阳性个体持续继代,工作量较大。因此急需获得遗传稳定、滞育性易受控制的注射材料。而滞育必需基因可作为我们创制滞育可调节的注射用家蚕材料的分子靶标。
此外,家蚕是重要的鳞翅目模式昆虫,滞育是鳞翅目农林害虫中普遍存在的特征,研究家蚕滞育相关基因,鉴定鳞翅目昆虫保守的滞育调节基因,以实现通过控制昆虫的滞育达到害虫防治的目标。
因此,急需鉴定家蚕家蚕滞育必需基因。为家蚕胚胎滞育的研究提供新的理解;为创制滞育可调节的家蚕材料提供理论基础;为鳞翅目害虫防控研究提供潜在的分子靶标。
发明内容
有鉴于此,本发明的目的之一在于提供一种家蚕卵滞育必需基因Bmtret1,本发明的目的之二在于提供所述家蚕卵滞育必需基因Bmtret1在调控家蚕卵滞育中的应用,本发明的目的之三在于提供一种抑制家蚕卵滞育的方法,本发明的目的之四在于提供一种含有抑制家蚕卵滞育必需基因Bmtret1的转录、翻译或功能的试剂或化合物。
为达到上述目的,本发明提供如下技术方案:
1、一个家蚕卵滞育必需基因Bmtret1,所述基因的核苷酸序列包括1)和2):
1)SEQ ID NO.1;
2)与SEQ ID NO.1至少有70%的碱基相同,且与该基因序列表达相同功能蛋白质的核苷酸序列。
作为优选的技术特征之一,所述基因编码蛋白的氨基酸序列包括1)和2):
1)SEQ ID NO.2所示;
2)将SEQ ID NO.2氨基酸序列经过一个或多个氨基酸残基的取代、缺失或添加而形成的且具有与该蛋白相同功能的衍生蛋白;或与SEQ ID NO.2氨基酸序列有至少50%的同源性且具有与该蛋白相同功能的衍生蛋白。
2、所述家蚕卵滞育必需基因Bmtret1在调控家蚕卵滞育中的应用。
3、一种抑制家蚕卵滞育的方法,所述方法为,以所述家蚕卵滞育必需基因Bmtret1为靶标,降低或抑制家蚕卵滞育必需基因Bmtret1的转录及翻译。
作为优选的技术特征之一,所述降低或抑制的方法包括但不限于基因突变、基因沉默和基因敲除。
作为优选的技术特征之一,所述基因敲除的方法为,采用CRISPR/Cas9系统进行基因编辑,敲除Bmtret1基因。
作为优选的技术特征之一,所述基因编辑的方法为,根据Bmtret1基因的cDNA序列,选择sgRNA结合位点,合成sgRNA序列,将sgRNA和Cas9蛋白混合后注入刚产的蚕卵中。
作为优选的技术特征之一,所述sgRNA结合位点序列为SEQ ID NO:3、SEQ ID NO:4。
4、含有抑制家蚕卵滞育必需基因Bmtret1的转录、翻译或功能的试剂或化合物。
本发明的有益效果在于:
本发明通过靶向家蚕Bmtret1基因,降低其表达量,或者破坏Bmtret1基因结构,可以使本应进入滞育的蚕卵转变为非滞育卵。这一发明可以用于在蚕业生产中培育滞育性可调节的家蚕材料和品种,同时为通过调控鳞翅目昆虫的卵滞育实现害虫防治提供一种新的思路。
附图说明
为了使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明作优选的详细描述,其中:
图1:家蚕Bmtret1基因的结构示意图及其sgRNA靶点(TS)序列。
图2:CRISPR/Cas9诱导的基因突变结果。
图3:Bmtret1基因敲除的Dazao纯合品系在25℃催青,子代卵不进入滞育。
具体实施方式
下面结合具体的优选实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。实施例中未注明具体条件的实验方法,通常按照常规条件如分子克隆实验指南(第三版,J.萨姆布鲁克等著)中所述的条件,或按照制造厂商所建议的条件。
实施例1
sgRNA靶点(TS)的选择及sgRNA的合成
(1)sgRNA结合位点的选择
本发明利用CRISPR/Cas9系统敲除Bmtret1基因,针对Bmtret1基因的序列、经过大量的选择及试验,利用CRISPRdirect网站(http://crispr.dbcls.jp/)依据GN19NGG的规则,在Bmtret1基因的第一外显子上选取了两个特异较高的sgRNA位点序列。Bmtret1基因的核苷酸序列如SEQ ID NO.1所示,氨基酸序列如SEQ ID NO.2所示。家蚕Bmtret1基因结构示意图及sgRNA靶点序列如图1所示。
sgRNA识别靶点序列如下:
sgRNA1:5’-AAAGAGGTAGTGATGACGAA-3’(SEQ ID NO:3)
sgRNA2:5’-GATTCGGATACAGGGATCGC-3’(SEQ ID NO:4)
(2)sgRNA的合成
①根据选择的sgRNA靶点序列,合成如下引物:
sgRNA-F1:
TAATACGACTCACTATAGGGAAAGAGGTAGTGATGACGAAGTTTTAGAGCTAGAAATAGC(SEQ IDNO:5)
sgRNA-F2:
TAATACGACTCACTATAGGGGATTCGGATACAGGGATCGCGTTTTAGAGCTAGAAATAGC(SEQ IDNO:6)
sgRNA-R:
AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTT
GCTATTTCTAGCTCTAAAACCCGGCCGCCCCGACTTACCACGTTAGGAAAATTAATA(SEQ ID NO:7)
②使用PrimeSTAR GXL高保真聚合酶进行退火延伸,PCR完成后,进行胶回收并纯化。
③以PCR回收纯化产物为模板,利用TranscriptAid T7 High YieldTranscription Kit试剂盒(购自Thermo Scientific公司)体外转录合成sgRNA并进行纯化。
④sgRNA的框架序列:
NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT(SEQ ID NO:8)。
实施例2
家蚕胚胎显微注射
①蚕卵制备:将已交配5h的低温催青大造雌蛾(产非滞育卵)拆对后轻轻放置于糊有浆糊的产卵纸上产卵,同时,严格把控产卵时间,使用产下时间在2h以内的蚕卵进行显微注射;
②蚕卵消毒:将新鲜蚕卵整齐粘在事先经过酒精消毒处理的载玻片上,并放入甲醛环境中消毒4min;
③蚕卵显微注射:将sgRNA和Cas9蛋白以2:1比例混匀,使gRNA终浓度1000ng/μL,Cas9终浓度为500ng/μL,在PCR仪中37℃反应15min后,用显微注射仪注射到刚产的蚕卵中;
④蚕卵封口与消毒:用适量胶水对注射后的蚕卵进行封口,待注射口被封住后,将蚕卵置于甲醛环境中进行消毒处理;
⑤蚕卵催青与保湿:将注射后的蚕卵放置于人工气候箱中催青(催青条件为25℃,70-80%的湿度),蚕卵孵育期间注意对注射蚕卵进行通风处理以防霉变,随时观察蚕卵发育情况。
实施例3
Bmtret1基因敲除纯合系筛选
精心饲养注射蚕卵材料至化蛾,并剪取蚕蛾翅膀提取基因组DNA,针对Bmtret1基因的sgRNA1和sgRNA2的序列,我们设计了特异性的检测引物用于对出蚁的G1代个体进行Bmtret1基因的DNA序列的分子克隆分析,检测引物为:
Bmtret1-TS-R:5’-TTAGAGACAAAATGAGATCGTGC-3’(SEQ ID NO.9)
Bmtret1-TS-F:5’-ATTCTAATCGTAATGTTCGGAAG-3’(SEQ ID NO.10)
将扩增获得的相应的基因组区段克隆到-T5 Zero载体上,挑单克隆测序,通过序列比对检测是否存在变异并确定变异的类型。筛选被编辑后该基因功能丧失的蚕蛾进行交配,继续饲养几代筛选Bmtret1基因敲除纯合系家蚕,CRISPR/Cas9诱导的基因突变如图2所示。
实施例4
Bmtret1基因敲除纯合系家蚕子代滞育性观察
将筛选获得的Bmtret1基因敲除的纯合品系家蚕卵在25℃条件下催青,观察子代卵是否进入滞育。结果如图3所示,结果表明下一代卵不再进入滞育。
本发明实施例1—4中以CRISPR/Cas9介导的基因编辑技术敲除Bmtret1基因,本领域技术人员公知,只要能够敲除或敲低Bmtret1基因的任何手段均可实现本发明的发明目的。本领域技术人员应当理解,可以对本发明作出各种修改,而不偏离本发明权利要求书所限定的范围。
序列表
<110> 西南大学
<120> 家蚕卵滞育的必需基因Bmtret1及其应用
<160> 10
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atgagatcgt gcttatttcg aaaaagaggt agtgatgacg aaagggaacc tttgcttccc 60
gcgatccctg tatccgaatc aagttcaaat accggaaatg agtgtgctag cgagacaaac 120
acaaacggca ggatggccaa tctcggtgtc tctcaaacag aactggttgg tcctcgggga 180
gattctggtc gcaagttacc tcaatacata gctgctttag cagctaccct tggagcacta 240
gctgctggaa caatgcttgg ttggtcttcg ccagttgtgt tcaaaataac acaaccaaac 300
aatacagact acaattttga catcagtgaa actgaaggaa gctggattgg gtctgtcatc 360
aacttgggtg ctgctgcaat ttgttttcca attggcctgg tcatggatgc tattggacga 420
aaaaaaacta tgctgtttct catcctcccg ttcactttgg gctggcttct tataactttt 480
ggtacgagtg tcggtatgct cataggcgga agactcatta ccggtattgc aggaggagct 540
ttctgtgtca ccgcaccggc atacactagt gaaatagccc aagactccat cagaggtacc 600
ctagggagct acttccaact gatgatcaca gttggtattc tatttgctta tgcagtgggt 660
agttacacat cagtgttcct tttcaatatt ctttgcactc tgatacctat tgtctttggt 720
attgtgttct ttttcatgcc tgaaagtcca aaatttttgg ttgtaaaaaa cagaaacgat 780
gaagctcgag aagcactaat taaactacgc ggtaccaatt acgatgttga ctacgagctg 840
gactctttga aactgagcgc tgaagaagca cagaacaatc cagtttcatt tgtttcggct 900
attacgaaga aaacgtctat taaagcgata ataatttgtt acgcactgat gatattccaa 960
cagctctctg gtataaatgc ggtcattttt aacacctcat cgattttcgc tgctgccggt 1020
gccacaattc cggccgcgat cgcgaccatt atcatcggcg tcatccaagt aatcgccaca 1080
tttgtatcca gtttggttgt cgacaaatta ggccgacgta ttttgcttct cttctccgct 1140
ttggttatgt gcttgtgttc gacggctttg ggggtgtttt tcttcttaca aagtacgcac 1200
ggagaaaatt ctgatatagt tcaaagtctt ttctggttac ctcttttgtc tttgtctctt 1260
ttcatcatcg cgttctctct tggtttcggt ccgatcccgt ggatgatggc tggcgatctc 1320
tgcaatattg acataaaggc gttcgtcggt tccactgccg ggacattgaa ctggctgctt 1380
agttttacgg tgaccagcac cttccttgcc ttgaatactg ctattggatc tggtcaggta 1440
ttctggatgt tcgcaggcat catgttgatt ggttttgtat tcattttctt cgtaataccg 1500
gagacaaaag gcaagagcct tcaagaaatt caagtgatgt taggagcgac accacaagat 1560
agaaacgtag aagacaaaaa gtga 1584
<210> 2
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Claims (5)
1.一种抑制家蚕卵滞育的方法,其特征在于,所述方法为,以家蚕卵滞育必需基因Bmtret1为靶标,降低或抑制家蚕卵滞育必需基因Bmtret1的转录及翻译,所述Bmtret1基因的核苷酸序列如SEQ ID NO.1所示。
2.如权利要求1所述的方法,其特征在于,所述降低或抑制的方法包括基因突变、基因沉默和基因敲除。
3.如权利要求2所述的方法,其特征在于,所述基因敲除的方法为,采用CRISPR/Cas9系统进行基因编辑,敲除Bmtret1基因。
4.如权利要求3所述的方法,其特征在于,所述基因编辑的方法为,根据Bmtret1基因的cDNA序列,选择sgRNA结合位点,合成sgRNA序列,将sgRNA和Cas9蛋白混合后注入刚产的蚕卵中。
5.如权利要求4所述的方法,其特征在于,所述sgRNA结合位点的核苷酸序列为SEQIDNO:3或SEQ ID NO:4。
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