CN113897370A - 家蚕BmTRPM基因及应用 - Google Patents

家蚕BmTRPM基因及应用 Download PDF

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CN113897370A
CN113897370A CN202111266980.6A CN202111266980A CN113897370A CN 113897370 A CN113897370 A CN 113897370A CN 202111266980 A CN202111266980 A CN 202111266980A CN 113897370 A CN113897370 A CN 113897370A
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时桂芹
魏涛
任菲
张书铭
周园
程军权
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Zhengzhou University of Light Industry
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Abstract

本发明提出了家蚕BmTRPM基因及应用，本发明的第一目的在于提供家蚕瞬时感受器电位通道调控基因BmTRPM；第二目的在于提供所述的家蚕瞬时感受器电位通道调控基因BmTRPM表达的蛋白。本发明将家蚕BmTRPM基因被抑制表达后会导致家蚕滞育的解除率明显升高，蛹期家蚕高温处理后导致家蚕滞育的解除率更高，可以达到33.5%。本发明通过抑制家蚕BmTRPM基因的表达，可实现快速、绿色阻止蚕卵滞育的目的，对于解决由于化学滞育解除剂导致的生态恶化以及解除时间长等问题具有积极意义。

Description

家蚕BmTRPM基因及应用
技术领域
本发明涉及属于基因工程技术领域，具体而言，涉及家蚕BmTRPM基因及应用。
背景技术
滞育是昆虫适应环境的重要生理特性，家蚕的滞育还与其经济性状有着密切关系，相关生物学问题一直是研究热点。而家蚕（Bombyx mori）属于鳞翅目（Lepidoptera）蚕蛾科（Bombycidae）昆虫，是我国主要的经济昆虫，在卵（胚胎）期滞育。二化性家蚕卵的滞育受母代胚胎期所处环境条件影响，然而其分子机制还没有得到深度的解析。对于进入滞育的蚕卵，需要人工打破滞育才能使其继续发育。在家蚕的蚕种生产实践中，家蚕滞育的解除通常采用低温冷藏或浸酸的方法。低温冷藏即将产下的滞育卵放在5℃条件下冷藏 2-3 个月来解除滞育；浸酸包括冷藏浸酸和即时浸酸两种方法，冷藏浸酸即滞育卵冷藏一段时间后通过浸酸处理解除滞育，即时浸酸即滞育卵产下 20h 通过浸酸阻止滞育的发动。滞育解除时还会用到的 H2O2 和二甲亚砜来解除家蚕的滞育。滞育卵产后 24h 用 H2O2处理，可以阻止滞育的起始。二甲亚砜可以有效地阻止家蚕卵进入滞育。在二甲亚砜的最佳处理浓度（100%）下，其处理效果可以与HCl处理解除滞育相当或接近。
瞬时受体电位通道（Transient receptor potential channel, TRP channel）是由存在于细胞膜上的一类重要阳离子通道构成的蛋白超家族。TRP通道在脊椎动物和非脊椎动物有机体中对温度变化的感知都发挥了重要的作用。家蚕中只有对BmTRPA1基因的少量相关报道，但对于家蚕中BmTRPM基因功能的研究还未见报道。
发明内容
本发明正是基于上述技术问题至少之一，本发明将家蚕BmTRPM基因被抑制表达后会导致家蚕滞育的解除率明显升高，蛹期家蚕高温处理后导致家蚕滞育的解除率更高，可以达到33.5%。本发明对于解决由于化学滞育解除剂导致的生态恶化以及低温解除滞育所需时间长等问题具有积极意义。
有鉴于此，根据本发明的第一个目的提出了一种家蚕BmTRPM基因，所述BmTRPM基因为所述家蚕瞬时感受器电位通道调控基因，核苷酸序列为SEQ ID No.1。
根据本发明的第二个目的提出了家蚕BmTRPM基因编码的蛋白，所述蛋白的氨基酸序列为SEQ ID No.2。
根据本发明的第三个目的提出了上述的家蚕BmTRPM基因或上述的家蚕BmTRPM基因编码的蛋白在解除家蚕滞育中的应用。
在一些实施例中，抑制家蚕BmTRPM基因的表达，提高家蚕滞育的解除率。
在一些实施例中，抑制家蚕BmTRPM基因的表达，同时27-30℃高温处理蛹期家蚕提高家蚕滞育的解除率。
在一些实施例中，以家蚕总RNA为模板扩增dsRNA沉默BmTRPM基因；所述dsRNA核苷酸序列为SEQ ID No.3。
在一些实施例中，扩增dsRNA的引物序列为SEQ ID No.4- SEQ ID No.5；
正向引物：5’-GTAATACGACTCACTATAGGGAGAGAACAGTTGTCTTTA
GCCCTCA -3’；SEQ ID No. 4；
反向引物：5’- GTAATACGACTCACTATAGGGAGAGAGAGTGTCTGTGGACC
CCC -3’；SEQ ID No. 5。
其中划线部分为T7启动子序列。
本发明首次提出了家蚕瞬时受体电位通道调控基因BmTRPM在用于家蚕BmTRPM基因被抑制表达后会导致家蚕滞育的解除率明显升高，蛹期家蚕高温处理后导致家蚕滞育的解除率更高，可以达到33.5%。对于解决由于化学滞育解除剂导致的生态恶化以及低温解除滞育所需时间长等问题具有积极意义。
通过以上技术方案，本发明提出了家蚕BmTRPM基因、蛋白质以及应用，本发明利用同源克隆技术从家蚕中得到瞬时受体电位通道调控基因BmTRPM，首次提出利用BmTRPM基因序列扩增的dsTRPM，抑制家蚕瞬时受体电位通道调控基因BmTRPM的表达：导致家蚕滞育的解除率明显升高，蛹期家蚕高温处理后导致家蚕滞育的解除率更高，可以达到33.5%。
附图说明
图1为本发明实施例1中BmTRPM基因的琼脂糖凝胶电泳结果；
其中，M为DNA Marker，1为目的片段。
图2为本发明实施例2中BmTRPM基因 dsRNA注射到家蚕体内后，BmTRPM基因在24h的表达量。
图3为本发明实施例3中将BmTRPM基因 dsRNA注射到家蚕体内后对家蚕滞育率的统计。
图4为本发明实施例3中将BmTRPM基因 dsRNA注射到家蚕体内后对家蚕滞育率的影响效果图，A：26℃（正常温度）饲养对照组；B：20℃（低温）处理dsTRPM实验组；C：26℃（正常温度）处理dsTRPM实验组；D：30℃（高温）处理dsTRPM实验组。
具体实施方式
为了能够更清楚地理解本发明的上述目的、特征和优点，下面结合附图和具体实施方式对本发明进行进一步的详细描述。需要说明的是，在不冲突的情况下，本发明的实施例及实施例中的特征可以相互组合。
在下面的描述中阐述了很多具体细节以便于充分理解本发明，但是，本发明还可以采用其他不同于在此描述的其他方式来实施，因此，本发明的保护范围并不受下面公开的具体实施例的限制。
实施例1
家蚕总RNA的提取和c DNA合成
（1）将-80℃超低温保存的家蚕整蚕取出，称0.1g后置于液氮中研磨，加入1m LTRIzol，用匀浆仪进行匀浆处理。将匀浆样品在室温放置5min，4℃，10 000g离心10min；小心取上清液移入新的Eppendorf管中，加入0.2m L氯仿，剧烈振荡15s，室温放置3min，4℃，10 000g离心10min；取上层澄清水相，转移到新的Eppendorf管中，加入0.5m L异丙醇，室温放置10min，4℃，10 000g离心10min；弃上清液；加入1m L 75%乙醇洗涤RNA沉淀；4℃，7500g离心5min，弃上清；将EP管倒置在超净工作台10-15min，加入无RNA free水，将所获得的RNA进行凝胶电泳分析，用分光光度计检测浓度和纯度。
（2）在冰上按下列反应组分配制反应液：
名称 体积(μL)
MgCl<sub>2</sub> (25mM) 4
10×RNA PCR buffer 2
dNTP Mixture (10 μM) 2
RNase Inhibitor (40U/μL) 0.5
ReverTra Ace 1
Oligo (dT)20 1
Total RNA (<1μg) 1
RNase Free dH<sub>2</sub>O 8.5
Total 20
在PCR仪上按以下程序进行反应：42℃，30min；95℃，5min；5℃，5min。反应结束后保存于4℃冰箱。
实施例2
家蚕BmTRPM基因dsRNA的合成和显微注射
根据家蚕BmTRPM基因序列SEQ ID NO.1，设计BmTRPM基因的dsRNA引物对，
扩增dsRNA的引物序列为SEQ ID No.4- SEQ ID No.5；
正向引物：5’-GTAATACGACTCACTATAGGGAGAGAACAGTTGTCTTTA
GCCCTCA -3’；SEQ ID No. 4；
反向引物：5’- GTAATACGACTCACTATAGGGAGAGAGAGTGTCTGTGGACC
CCC -3’；SEQ ID No. 5。
通过体外转录试剂合成dsRNA，dsRNA序列为SEQ ID No.3，具体地，通过RiboMAXLarge Scale RNA Production System-T7试剂盒，按照以下的20 μL体系体外合成dsRNA。
RiboMAXTM Express T7 2×Buffer 10 μL
Enzyme Mix 2 μL
cDNA 3 μL
Nuclease-Free Water up to 20 μL
将上述溶液加入1.5 ml离心管中混匀，放入37℃培养箱培养4-6 h。于70℃水浴中反应10 min，慢慢冷却至室温，冷却过程大约为20min。RNase Solution 稀释200倍。每20 μL体系加入上述1 μL稀释过的溶液以及1 μL RQ1 RNase-Free DNase，37℃孵育30 min。以总体积：3M Sodiam Acetate =10:1的比例以及总体积：无水乙醇=1:2.5的比例加入上述溶液，混匀，与-20℃中反应1-2h。12000 rpm，4℃离心10 min，弃上清。在沉淀中加入0.5ml预冷的浓度为70%的乙醇溶液，反复漂洗沉淀。12000 rpm，4℃离心10 min，除去乙醇，室温风干约15 min。加入无RNase的水溶解，悬浮沉淀。稀释后电泳，并测量浓度。
选取5龄2天、大小均一、健康状况一致的家蚕幼虫，设置注射的dsGFP对照组和注射dsTRPM的实验组，注射前将家蚕幼虫在冰上麻醉，然后利用微量注射器将体外合成的dsRNA注射至家蚕幼虫的体腔，dsRNA的注射量为10 μg/头，每组注射60头，设置3个生物学重复，注射完毕后，将家蚕幼虫置于人工气候箱内饲养。
利用荧光定量PCR检测显微注射后24h时BmTRPM基因的表达，效果如图2，由图2可知，发现注射24h后能够高效沉默家蚕BmTRPM基因的表达，具有较好的靶基因沉默效果。
实施例3
家蚕BmTRPM基因的dsRNA会导致家蚕滞育的阻止率明显升高，蛹期家蚕高温处理后导致家蚕滞育的阻止率更高。
待注射dsTRPM的实验组家蚕完全结茧化蛹后，将其分别放到20℃（低温）、26℃（正常温度）和30℃（高温）人工气候箱内，分别处理48h，dsGFP对照组一直放到26℃（正常温度）下饲养。待蛹孵化蚕蛾进行交配，对不同温度处理的dsTRPM实验组产卵数进行统计，并对其中的非滞育卵的数目进行统计，每组统计四只母蛾的产卵量，并取其平均数如图3和图4所示。由图3和图4可知，注射dsRNA的处理组与对照组相比，其滞育率明显减少，蛹期家蚕高温处理后导致家蚕滞育率更低。表明家蚕BmTRPM基因的表达被抑制后，家蚕的滞育率大大降低。
可见，本发明基于分子层面降低使用家蚕滞育解除化学试剂的设计方法，对于提高家蚕滞育解除效率，防治生态环境污染，同时也对增强其它鳞翅目害虫的防治具有重要意义。
以上仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 郑州轻工业大学
<120> 家蚕BmTRPM基因及应用
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3497
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggcgttac tcttaccgat aacggtcttc aggattcaag agcgtgcaga aggtggagaa 60
gtccaagagc ttgatcaggt catactgacg gcgttgttcc gggcccagca tctgactcca 120
agtgaacagt tgtctttagc cctcacttgg aaccgggtcg acatcgccag atccgagatc 180
ttcgtttacg gacaggaatg gcctccaggc gccctagacg aagccatgat gcaagccctg 240
gagcacgata ggatcgactt cgttaagctt ctcctcgaga acggcgtgtc catgaggaaa 300
ttcctcacga ttcctagact tgaagaactt tacaacacga agagcgggcc tagtaacacc 360
ttgaggtaca tcttaaggga cgtgaggcct catctgccca aaggctacgt gtacacgctt 420
cacgacatcg gtttggtgat caacaaactc attggcggag cttataggtg ctactacaca 480
cgtcggaagt ttcgaccgat ctacgcaaag gtgatgaaca aaagcgtcaa tgtacatcgc 540
aacagtgcgt ccttcaccag gcataacgct ggaggcctgt cgctcatcac cgggttcatg 600
ccggtcacca gtgaaatggc gcttttcgat tatcctttca acgagctttt aatgtgggca 660
gtcctaacga agagacatca aatggcgctc ctcatgtgga cacatggcga ggagtcgctt 720
gcgaaatcgc tcatagcatg caagctgtac aaggccatgg cccacgaggc agccgaggat 780
gatatggaga ctgaagttta tgaagagcta aggcactacg gcaaagaatt tgagaataag 840
gctttggagc ttctagatta ctgctaccgc caagatgatg atcaggccca gcaactcctc 900
acgtgcgagc tccagaactg gtccggtcag acctgtctca gcttagccgt gaccgcgaac 960
catcgtgcgc tactcgctca tccgtgctca caaataatct tagcggactt atggatgggc 1020
ggcctcagaa cgagaaagaa caccaatctc aaggtaattc tgagcctgct gtgtccgctg 1080
tacattctga gattggagtt caagtccaaa gaggaacttc agctgatgcc gcagacggaa 1140
gaagagcatc tagagaacga gagcatcgag gatgatagga gcaccaagga tccgacagat 1200
gccgagggga atgcgagacc cgggtcgatc agaacgggaa agttggtaaa gtaggatggg 1260
aaccatcgta cagagagctg ccagagtacc acccatctga aattaaacga atgaggccac 1320
tgcgactgag gaagaagatc tatgagttct ttacggcacc gatcactaaa ttttgggccg 1380
attcgatagc ctacatattg ttcctgttga tgtttacata cacagtatta gtcaagatga 1440
atcctacacc ttcttggccg gaaatatatt ctatatgtta tatattaact ttcttatgcg 1500
agaagattag agaaatcgta acatccgagc ctgttgccat tagacataag ttcagtgtgt 1560
gggcatggaa catgtggaac acgtatgacg ctggttttat aatattcttc ttagttggat 1620
tgacgctgag actcagagag atctcaagag acgtagggag agttatatac tgcgtcgata 1680
ttatatattg gtacctaagg atcttgaata tattgggtgt caataagtac ttaggtcccc 1740
tagtcacaat gatgggtaaa atggtaaaga acatgatata cttcgtggtg ctattgctag 1800
tggtattaat gtctttcggt gttgccagac aagctatact ccatccagat aaggacgcta 1860
gttggcatct aatcagagag gtattctacc aaccgtactt catgttgtac ggtgaagtat 1920
tcgctggcga tatagatcct ccttgcggga aggagatcgg cgaccggaaa tgcgtcaccg 1980
gaagatggat cacgcccatc gccatgacag tgtacctgct catcgccaat atattactga 2040
tcaatctcct aattgccgtt tttaataata tattcaacga agtcaatgaa gtctcgcatc 2100
aggtgtggat gttccaaagg ttcacagtgg tcatggagta tgaacagaag ccagttctgc 2160
cgccaccgct gatcatcttc tgccacatct acgccgttgg caagtgggtg accagaaacg 2220
tctcgcacaa gagattccaa tacgacaacg gactgaagct gttcttggag aaagaagaca 2280
tggagaggct ttatgacttc gaagaggaat gcgtggaggg atactttagg gaacaagaaa 2340
taaaattatc gaattcaata gaggagagag ttaggaatac aacagatagg gtagaacata 2400
taacgaccaa aatggaagac ttgaatcaaa aggcaaattg ccaaatacaa tcgatgcaga 2460
gcgttgaatt taggttaaga aagctcgaag atatagcgga gcagacgatg agccaccagg 2520
cggtgataca caggttcatg gcgactcatc gcttcgctgg tcaaccgggg gtccacagac 2580
actctcaagg ccggttactc ccatcacaac acttggctcc gaggagacag cacagtcaaa 2640
cgcattctga acccgacaac tctgcagtag acgctggttc tgtgcacaat tccgtaacag 2700
gtcctcaatc ggggtgggag ggcgcggggg ccacgggggc cgcggggggg acagggaggg 2760
ctcgcgtggg gggcgcgccc cgctccctgc tgctcgccat gcacgagtac acgagcatca 2820
cggacgaact ggaaactgtt tatggactct tctcaccgcc gcacacgccg agaacaccga 2880
taacttcacg tctgctgtcc cccgcccgcg cggcctctcc ctccgtccgt cgacgtcacg 2940
ccagcgagat gtccaacccc gagttcgcac tcttcatgga gaaggagcac cttcgaggag 3000
ccgaagaaga cgactacatc atcatggaga acctgatcca aaggcgccta gaaatggggt 3060
ctgtactgga cagatacgag gaggaggagg gcggggaggg ggcgggcgcg gggggagcga 3120
tcagcatcag cgtctgcgtc aacaccagcg aggagcaacc acagagttcg agtctcctca 3180
ccgtgacaga cagacgtcta ccgcatcaga ggcactcgct aaggagatct tcagctgtcg 3240
acagccggga gcttccgtcg cccctgcagc ctctgctcgg agatgatgct tgttgcgaag 3300
tcttactgcc ggttccgaga cagccttcgg gagatacgaa cgcatctagc gacctctcgc 3360
tctcacacat ggtgagcgag aatctcccgc cgcggccctc catagtgctc gactcgtctc 3420
agctcccgcg tcaaaggtcc gcggagcaac agcagacgca gcctccggga ccgtccagac 3480
ccgagacaat gtgctaa 3497
<210> 2
<211> 1116
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ala Leu Leu Leu Pro Ile Thr Val Phe Arg Ile Gln Glu Arg Ala
1 5 10 15
Glu Gly Gly Glu Val Gln Glu Leu Asp Gln Val Ile Leu Thr Ala Leu
20 25 30
Phe Arg Ala Gln His Leu Thr Pro Ser Glu Gln Leu Ser Leu Ala Leu
35 40 45
Thr Trp Asn Arg Val Asp Ile Ala Arg Ser Glu Ile Phe Val Tyr Gly
50 55 60
Gln Glu Trp Pro Pro Gly Ala Leu Asp Glu Ala Met Met Gln Ala Leu
65 70 75 80
Glu His Asp Arg Ile Asp Phe Val Lys Leu Leu Leu Glu Asn Gly Val
85 90 95
Ser Met Arg Lys Phe Leu Thr Ile Pro Arg Leu Glu Glu Leu Tyr Asn
100 105 110
Thr Lys Ser Gly Pro Ser Asn Thr Leu Arg Tyr Ile Leu Arg Asp Val
115 120 125
Arg Pro His Leu Pro Lys Gly Tyr Val Tyr Thr Leu His Asp Ile Gly
130 135 140
Leu Val Ile Asn Lys Leu Ile Gly Gly Ala Tyr Arg Cys Tyr Tyr Thr
145 150 155 160
Arg Arg Lys Phe Arg Pro Ile Tyr Ala Lys Val Met Asn Lys Ser Val
165 170 175
Asn Val His Arg Asn Ser Ala Ser Phe Thr Arg His Asn Ala Gly Gly
180 185 190
Leu Ser Leu Ile Thr Gly Phe Met Pro Val Thr Ser Glu Met Ala Leu
195 200 205
Phe Asp Tyr Pro Phe Asn Glu Leu Leu Met Trp Ala Val Leu Thr Lys
210 215 220
Arg His Gln Met Ala Leu Leu Met Trp Thr His Gly Glu Glu Ser Leu
225 230 235 240
Ala Lys Ser Leu Ile Ala Cys Lys Leu Tyr Lys Ala Met Ala His Glu
245 250 255
Ala Ala Glu Asp Asp Met Glu Thr Glu Val Tyr Glu Glu Leu Arg His
260 265 270
Tyr Gly Lys Glu Phe Glu Asn Lys Ala Leu Glu Leu Leu Asp Tyr Cys
275 280 285
Tyr Arg Gln Asp Asp Asp Gln Ala Gln Gln Leu Leu Thr Cys Glu Leu
290 295 300
Gln Asn Trp Ser Gly Gln Thr Cys Leu Ser Leu Ala Val Thr Ala Asn
305 310 315 320
His Arg Ala Leu Leu Ala His Pro Cys Ser Gln Ile Ile Leu Ala Asp
325 330 335
Leu Trp Met Gly Gly Leu Arg Thr Arg Lys Asn Thr Asn Leu Lys Val
340 345 350
Ile Leu Ser Leu Leu Cys Pro Leu Tyr Ile Leu Arg Leu Glu Phe Lys
355 360 365
Ser Lys Glu Glu Leu Gln Leu Met Pro Gln Thr Glu Glu Glu His Leu
370 375 380
Glu Asn Glu Ser Ile Glu Asp Asp Arg Ser Thr Lys Asp Pro Thr Asp
385 390 395 400
Ala Glu Gly Asn Ala Arg Pro Gly Ser Ile Arg Thr Gly Lys Leu Val
405 410 415
Lys Asp Gly Asn His Arg Thr Glu Ser Cys Gln Ser Thr Thr His Leu
420 425 430
Lys Leu Asn Glu Gly His Cys Asp Gly Arg Arg Ser Met Ser Ser Leu
435 440 445
Arg His Arg Ser Leu Asn Phe Gly Pro Ile Arg Pro Thr Tyr Cys Ser
450 455 460
Cys Cys Leu His Thr Gln Tyr Ser Arg Ile Leu His Leu Leu Gly Arg
465 470 475 480
Lys Tyr Ile Leu Tyr Val Ile Tyr Leu Ser Tyr Ala Arg Arg Leu Glu
485 490 495
Lys Ser His Pro Ser Leu Leu Pro Leu Asp Ile Ser Ser Val Cys Gly
500 505 510
His Gly Thr Cys Gly Thr Arg Met Thr Leu Val Leu Tyr Ser Ser Leu
515 520 525
Asp Arg Asp Ser Glu Arg Ser Gln Glu Thr Gly Glu Leu Tyr Thr Ala
530 535 540
Ser Ile Leu Tyr Ile Gly Thr Gly Ser Ile Tyr Trp Val Ser Ile Ser
545 550 555 560
Thr Val Pro Ser Gln Trp Val Lys Trp Arg Thr Tyr Thr Ser Trp Cys
565 570 575
Tyr Cys Trp Tyr Cys Leu Ser Val Leu Pro Asp Lys Leu Tyr Ser Ile
580 585 590
Gln Ile Arg Thr Leu Val Gly Ile Ser Glu Arg Tyr Ser Thr Asn Arg
595 600 605
Thr Ser Cys Cys Thr Val Lys Tyr Ser Leu Ala Ile Ile Leu Leu Ala
610 615 620
Gly Arg Arg Ser Ala Thr Gly Asn Ala Ser Pro Glu Asp Gly Ser Arg
625 630 635 640
Pro Ser Pro Gln Cys Thr Cys Ser Ser Pro Ile Tyr Tyr Ser Ile Ser
645 650 655
Leu Pro Phe Leu Ile Ile Tyr Ser Thr Lys Ser Met Lys Ser Arg Ile
660 665 670
Arg Cys Gly Cys Ser Lys Gly Ser Gln Trp Ser Trp Ser Met Asn Arg
675 680 685
Ser Gln Phe Cys Arg His Arg Ser Ser Ser Ala Thr Ser Thr Pro Leu
690 695 700
Ala Ser Gly Pro Glu Thr Ser Arg Thr Arg Asp Ser Asn Thr Thr Thr
705 710 715 720
Asp Ser Cys Ser Trp Arg Lys Lys Thr Trp Arg Gly Phe Met Thr Ser
725 730 735
Lys Arg Asn Ala Trp Arg Asp Thr Leu Gly Asn Lys Lys Asn Tyr Arg
740 745 750
Ile Gln Arg Arg Glu Leu Gly Ile Gln Gln Ile Gly Asn Ile Arg Pro
755 760 765
Lys Trp Lys Thr Ile Lys Arg Gln Ile Ala Lys Tyr Asn Arg Cys Arg
770 775 780
Ala Leu Asn Leu Gly Glu Ser Ser Lys Ile Arg Ser Arg Arg Ala Thr
785 790 795 800
Arg Arg Tyr Thr Gly Ser Trp Arg Leu Ile Ala Ser Leu Val Asn Arg
805 810 815
Gly Ser Thr Asp Thr Leu Lys Ala Gly Tyr Ser His His Asn Thr Trp
820 825 830
Leu Arg Gly Asp Ser Thr Val Lys Arg Ile Leu Asn Pro Thr Thr Leu
835 840 845
Gln Thr Leu Val Leu Cys Thr Ile Pro Gln Val Leu Asn Arg Gly Gly
850 855 860
Arg Ala Arg Gly Pro Arg Gly Pro Arg Gly Gly Gln Gly Gly Leu Ala
865 870 875 880
Trp Gly Ala Arg Pro Ala Pro Cys Cys Ser Pro Cys Thr Ser Thr Arg
885 890 895
Ala Ser Arg Thr Asn Trp Lys Leu Phe Met Asp Ser Ser His Arg Arg
900 905 910
Thr Arg Arg Glu His Arg Leu His Val Cys Cys Pro Pro Pro Ala Arg
915 920 925
Pro Leu Pro Pro Ser Val Asp Val Thr Pro Ala Arg Cys Pro Thr Pro
930 935 940
Ser Ser His Ser Ser Trp Arg Arg Ser Thr Phe Glu Glu Pro Lys Lys
945 950 955 960
Thr Thr Thr Ser Ser Trp Arg Thr Ser Lys Gly Ala Lys Trp Gly Leu
965 970 975
Tyr Trp Thr Asp Thr Arg Arg Arg Arg Ala Gly Arg Gly Arg Ala Arg
980 985 990
Gly Glu Arg Ser Ala Ser Ala Ser Ala Ser Thr Pro Ala Arg Ser Asn
995 1000 1005
His Arg Val Arg Val Ser Ser Pro Gln Thr Asp Val Tyr Arg Ile Arg
1010 1015 1020
Gly Thr Arg Gly Asp Leu Gln Leu Ser Thr Ala Gly Ser Phe Arg Arg
1025 1030 1035 1040
Pro Cys Ser Leu Cys Ser Glu Met Met Leu Val Ala Lys Ser Tyr Cys
1045 1050 1055
Arg Phe Arg Asp Ser Leu Arg Glu Ile Arg Thr His Leu Ala Thr Ser
1060 1065 1070
Arg Ser His Thr Trp Ala Arg Ile Ser Arg Arg Gly Pro Pro Cys Ser
1075 1080 1085
Thr Arg Leu Ser Ser Arg Val Lys Gly Pro Arg Ser Asn Ser Arg Arg
1090 1095 1100
Ser Leu Arg Asp Arg Pro Asp Pro Arg Gln Cys Ala
1105 1110 1115
<210> 3
<211> 2480
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gaacagttgt ctttagccct cacttggaac cgggtcgaca tcgccagatc cgagatcttc 60
gtttacggac aggaatggcc tccaggcgcc ctagacgaag ccatgatgca agccctggag 120
cacgatagga tcgacttcgt taagcttctc ctcgagaacg gcgtgtccat gaggaaattc 180
ctcacgattc ctagacttga agaactttac aacacgaaga gcgggcctag taacaccttg 240
aggtacatct taagggacgt gaggcctcat ctgcccaaag gctacgtgta cacgcttcac 300
gacatcggtt tggtgatcaa caaactcatt ggcggagctt ataggtgcta ctacacacgt 360
cggaagtttc gaccgatcta cgcaaaggtg atgaacaaaa gcgtcaatgt acatcgcaac 420
agtgcgtcct tcaccaggca taacgctgga ggcctgtcgc tcatcaccgg gttcatgccg 480
gtcaccagtg aaatggcgct tttcgattat cctttcaacg agcttttaat gtgggcagtc 540
ctaacgaaga gacatcaaat ggcgctcctc atgtggacac atggcgagga gtcgcttgcg 600
aaatcgctca tagcatgcaa gctgtacaag gccatggccc acgaggcagc cgaggatgat 660
atggagactg aagtttatga agagctaagg cactacggca aagaatttga gaataaggct 720
ttggagcttc tagattactg ctaccgccaa gatgatgatc aggcccagca actcctcacg 780
tgcgagctcc agaactggtc cggtcagacc tgtctcagct tagccgtgac cgcgaaccat 840
cgtgcgctac tcgctcatcc gtgctcacaa ataatcttag cggacttatg gatgggcggc 900
ctcagaacga gaaagaacac caatctcaag gtaattctga gcctgctgtg tccgctgtac 960
attctgagat tggagttcaa gtccaaagag gaacttcagc tgatgccgca gacggaagaa 1020
gagcatctag agaacgagag catcgaggat gataggagca ccaaggatcc gacagatgcc 1080
gaggggaatg cgagacccgg gtcgatcaga acgggaaagt tggtaaagta ggatgggaac 1140
catcgtacag agagctgcca gagtaccacc catctgaaat taaacgaatg aggccactgc 1200
gactgaggaa gaagatctat gagttcttta cggcaccgat cactaaattt tgggccgatt 1260
cgatagccta catattgttc ctgttgatgt ttacatacac agtattagtc aagatgaatc 1320
ctacaccttc ttggccggaa atatattcta tatgttatat attaactttc ttatgcgaga 1380
agattagaga aatcgtaaca tccgagcctg ttgccattag acataagttc agtgtgtggg 1440
catggaacat gtggaacacg tatgacgctg gttttataat attcttctta gttggattga 1500
cgctgagact cagagagatc tcaagagacg tagggagagt tatatactgc gtcgatatta 1560
tatattggta cctaaggatc ttgaatatat tgggtgtcaa taagtactta ggtcccctag 1620
tcacaatgat gggtaaaatg gtaaagaaca tgatatactt cgtggtgcta ttgctagtgg 1680
tattaatgtc tttcggtgtt gccagacaag ctatactcca tccagataag gacgctagtt 1740
ggcatctaat cagagaggta ttctaccaac cgtacttcat gttgtacggt gaagtattcg 1800
ctggcgatat agatcctcct tgcgggaagg agatcggcga ccggaaatgc gtcaccggaa 1860
gatggatcac gcccatcgcc atgacagtgt acctgctcat cgccaatata ttactgatca 1920
atctcctaat tgccgttttt aataatatat tcaacgaagt caatgaagtc tcgcatcagg 1980
tgtggatgtt ccaaaggttc acagtggtca tggagtatga acagaagcca gttctgccgc 2040
caccgctgat catcttctgc cacatctacg ccgttggcaa gtgggtgacc agaaacgtct 2100
cgcacaagag attccaatac gacaacggac tgaagctgtt cttggagaaa gaagacatgg 2160
agaggcttta tgacttcgaa gaggaatgcg tggagggata ctttagggaa caagaaataa 2220
aattatcgaa ttcaatagag gagagagtta ggaatacaac agatagggta gaacatataa 2280
cgaccaaaat ggaagacttg aatcaaaagg caaattgcca aatacaatcg atgcagagcg 2340
ttgaatttag gttaagaaag ctcgaagata tagcggagca gacgatgagc caccaggcgg 2400
tgatacacag gttcatggcg actcatcgct tcgctggtca accgggggtc cacagacact 2460
ctcaaggccg gttactccca 2480
<210> 4
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gtaatacgac tcactatagg gagagaacag ttgtctttag ccctca 46
<210> 5
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gtaatacgac tcactatagg gagagagagt gtctgtggac cccc 44

Claims (7)

1.一种家蚕BmTRPM基因，其特征在于，所述BmTRPM基因为所述家蚕瞬时感受器电位通道调控基因，核苷酸序列为SEQ ID No.1。
2.权利要求1所述的家蚕BmTRPM基因编码的蛋白，其特征在于，所述蛋白的氨基酸序列为SEQ ID No.2。
3.如权利要求1所述的家蚕BmTRPM基因或如权利要求2所述的家蚕BmTRPM基因编码的所述蛋白在解除家蚕滞育中的应用。
4.权利要求3所述的应用，其特征在于，抑制家蚕BmTRPM基因的表达，提高家蚕滞育的解除率。
5.权利要求3或4所述的应用，其特征在于，抑制家蚕BmTRPM基因的表达，同时27-30℃处理蛹期家蚕提高家蚕滞育的解除率。
6.根据权利要求5所述的应用，其特征在于，以家蚕总RNA为模板扩增dsRNA沉默BmTRPM基因；所述dsRNA核苷酸序列为SEQ ID No.3。
7.根据权利要求5所述的应用，其特征在于，扩增dsRNA的引物序列为SEQ ID No.4-SEQ ID No.5；
正向引物：5’-GTAATACGACTCACTATAGGGAGAGAACAGTTGTCTTTA
GCCCTCA -3’；SEQ ID No. 4；
反向引物：5’- GTAATACGACTCACTATAGGGAGAGAGAGTGTCTGTGGACC
CCC -3’；SEQ ID No. 5。
CN202111266980.6A 2021-10-29 2021-10-29 家蚕BmTRPM基因及应用 Pending CN113897370A (zh)

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