阅读说明：本技术 烟粉虱MED隐种可溶性海藻糖酶及其基因BtTreh1和应用 (Bemisia tabaci MED cryptophyte soluble trehalase, gene BtTreh1 and application thereof ) 是由 吕志创 申晓娜 冀顺霞 王晓迪 郭建英 刘万学 万方浩 于 2019-10-26 设计创作，主要内容包括：本发明属于农业生物技术领域,具体涉及烟粉虱MED隐种可溶性海藻糖酶及其基因BtTreh1和应用。本发明的烟粉虱MED隐种可溶性海藻糖酶,其氨基酸序列如SEQ ID NO.1所示。本发明明确BtTreh1基因在烟粉虱MED温度耐受性中的作用,为进一步研究烟粉虱MED隐种的温度耐受性机理与抗寒关键物质海藻糖之间的关系及研究控制烟粉虱危害及扩散的方法提供依据。(The invention belongs to the technical field of agricultural biology, and particularly relates to bemisia tabaci MED cryptomorphic soluble trehalase, a gene BtTreh1 and application thereof. The amino acid sequence of the bemisia tabaci MED cryptophyte soluble trehalase is shown in SEQ ID No. 1. The invention defines the function of the BtTreh1 gene in the temperature tolerance of Bemisia tabaci MED, and provides a basis for further researching the relationship between the temperature tolerance mechanism of the hidden species of the Bemisia tabaci MED and trehalose which is a key cold-resistant substance, and researching a method for controlling the harm and the diffusion of the Bemisia tabaci.)

1. The bemisia tabaci MED cryptophyte soluble trehalase is characterized in that the amino acid sequence of the bemisia tabaci MED cryptophyte soluble trehalase is shown as SEQ ID No. 1.

2. A Bemisia tabaci MED cryptophyte soluble trehalase gene BtTreh1, encoding the Bemisia tabaci MED cryptophyte soluble trehalase of claim 1.

3. The bemisia MED cryptic soluble trehalase gene BtTreh1 of claim 2, wherein the nucleotide sequence is shown in SEQ ID No. 2.

4. A recombinant expression vector comprising the Bemisia tabaci MED cryptic soluble trehalase gene BtTreh1 of claim 2.

5. A recombinant strain comprising the bemisia tabaci MED cryptophyte soluble trehalase gene BtTreh1 of claim 2.

6. The application of the Bemisia tabaci MED cryptic soluble trehalase gene BtTreh1 in claim 2.

7. The application of the bemisia tabaci MED cryptophysoxidase gene BtTreh1 in preventing and controlling bemisia tabaci.

8. A method of reducing temperature tolerance of bemisia tabaci comprising the step of feeding the dsRNA of the bemisia tabaci MED cryptic soluble trehalase gene BtTreh1 of claim 2.

9. The method of reducing temperature tolerance of bemisia tabaci according to claim 8, wherein the dsRNA is amplified from the following primers:

BtTreh1-F：5’-TAATACGACTCACTATAGGGTCACCTGCCTTTTCCAC-3’，

BtTreh1-R：5’-TCCGATATTATGCTCCC-3’。

10. an agent for controlling bemisia tabaci, comprising the dsRNA fragment of the bemisia tabaci MED cryptic soluble trehalase gene BtTreh1 of claim 2.

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to bemisia tabaci MED cryptomorphic soluble trehalase, a gene BtTreh1 and application thereof.

Background

Bemisia tabaci (Gennadius)) belongs to the phylum Arthropoda, Insecta, Hemiptera, Bemisia, also known as Bemisia gossypii or Bemisia batatas. The most suitable development temperature of the bemisia tabaci is 26-30 ℃, the development critical temperature is 10.8-12.5 ℃, and the lethal high-temperature area is 37-42 ℃. 17 ℃ and 35 ℃ are the lowest and highest temperature limits for normal growth and development of bemisia tabaci. The strong temperature stress adaptability is the reason that bemisia tabaci is widely distributed in the world. With the increase of greenhouse effect, the global temperature increases year by year, and the tobacco whitefly MED has stronger temperature adversity adaptability to enable the tobacco whitefly MED to successfully adapt to different geographic environments, which has widely led people to discuss the theory of the invasion mechanism, and is one of the research hotspots in recent years for the molecular mechanism of the temperature adaptability.

The investigation on the distribution time and range of the mealybug MED species in China discovers that the invasion causes that the short fifteen years of China have spread to most provinces and cities, and the strong temperature adaptability of the mealybug MED species is greatly related to the content of stress-resistant substances in the mealybug MED species.

The phenomenon of RNA interference (RNAi) is an evolutionarily conserved defense mechanism against transgene or foreign virus invasion. Introduction of double-stranded RNA (dsRNA) having a sequence homologous to the mRNA of the transcription product of a target gene into a cell specifically degrades the mRNA, resulting in a corresponding loss of functional phenotype. RNAi is widely existed in biology world, and can silence some genes in insect body by RNAi technology to enhance or lose some abilities of insect, and also can inhibit the expression of functional gene in specific time to make the development of insect stay at a certain stage, so as to achieve the purpose of utilizing or preventing the damage of insect. The dsRNA is fed to the bemisia tabaci, so that the dsRNA has the characteristics of simplicity, convenience, easiness in operation and the like, and can be applied to the research of the bemisia tabaci.

Disclosure of Invention

The invention aims to provide a soluble trehalase of Bemisia tabaci MED cryptic species.

The invention further aims to provide a coding gene BtTreh1 of the bemisia tabaci MED cryptic soluble trehalase.

It is still another object of the present invention to provide a recombinant expression vector containing the above-mentioned coding gene.

It is still another object of the present invention to provide a recombinant strain containing the above-mentioned encoding gene.

It is still another object of the present invention to provide the use of the above-mentioned encoding gene.

According to the specific embodiment of the invention, the amino acid sequence of the bemisia tabaci MED cryptic soluble trehalase is shown as SEQID NO. 1:

MLWQTFPHLLARSANLLLSDQSKAGHNLHIFRLQLLSDHLFWPRRRVNRNNHYFAAGRRKAAALNAVCLSTSLDYNLIDDYNTNYLPSCYSKIYCDSELLHDVELAHIYPDSKTFVDKRMKFSEPYILSKYEELKAKNEGKPPSKEELVDFVSEHFEDGDELEKWDPPDFKPETTLMAKVSDEGYKKFLSGLQQVWKILARKIKPEVNENSDRYSLIYVPNGFCIPGGRFRELYYWDTYWIINGLLLSDMNDTAKGIIENLLSLVQKIGFIPNGSRVYYLNRSQPPLLIQMMNNYYKATNDFQFIKKNIKTLTKEFEWWQTNRKVKFIKDKKTYNMFRYYAPSNGPRPESYREDYEIAQTLPSESERTRWYTRIKSAAESGWDFSSRWFIKDGAGNGTLLDVHTPSIIPVDLNAFLHKNAVLLSEWWYMMGDKYRGKYFKEIAEKLLASINEVLWNENIGSWFDYDLINKQHRKYFFPSNIAPLWTESYSQPKNFMAAKVIEYIKREKIIKDDYTVHYHGIPSSLERTGQQWDFPNAWAPVQVFLIQGLDRTNVPQAQAIALKLAQDWVHSNYLGFQKTGFMYEKYNVEKAGDNGGGGEYESQISFGWSNGVVFEMMDRYATGLSSATLTR

the amino acid sequence has the typical structural characteristics of the Treh1 protein: comprises two tag sequences and a glycine-rich region, wherein the first tag sequence "PGGRFRELYYWDTY" is located at position 226-reservoir 239, the second tag sequence "QWDFPNAWAP" is located at position 531-reservoir 540, and the glycine-rich region is located at position 595-reservoir 600.

The coding gene BtTreh1 of the bemisia tabaci MED cryptic soluble trehalase has the genome sequence shown in SEQ ID NO. 2:

GCTTTTTCTCACCATATGTAGGAAGTGCATAAGTTCAAGCTCCAAGGTTGCGATTCCGAAATTTATTGATGAAGTTTTTGTAAACTCCTATTTTGTTCTCTTCTTCCCTCTCAGCATTCAAACAACTCAAACCAAAGTGTACTAGCTCTTGTTTCTTCCCGTCCTCATCGTTTTTCATTCAGCAGATTCAATTCCTTGAAGTTCATCCTAAACTCAGTGTCCAATCACACTTTAAAAGAATGTTGTGGCAGACCTTCCCACACTTACTGGCCCGAAGTGCTAATCTACTTCTCAGCGATCAAAGCAAAGCCGGCCACAATTTGCACATCTTTCGGCTTCAACTCCTGTCAGACCACCTGTTTTGGCCACGCAGGCGAGTGAATAGGAACAATCACTATTTTGCAGCCGGAAGGAGAAAAGCAGCCGCATTAAATGCAGTTTGCTTATCAACAAGCCTTGATTACAATCTTATTGATGATTATAATACTAACTACTTACCCTCTTGTTACAGCAAAATCTACTGCGACAGTGAACTCTTGCATGATGTTGAACTGGCCCACATCTATCCTGACTCAAAGACCTTTGTGGACAAGCGGATGAAATTTTCTGAACCCTATATTCTGTCCAAATATGAAGAACTAAAGGCTAAGAATGAGGGAAAACCGCCTTCAAAAGAAGAGCTTGTCGATTTTGTCTCAGAGCACTTTGAAGATGGAGATGAACTCGAAAAGTGGGACCCGCCTGATTTTAAACCTGAAACAACTCTCATGGCCAAAGTTTCGGATGAAGGTTACAAAAAATTCCTGAGTGGACTACAACAAGTTTGGAAAATTCTAGCCCGAAAGATCAAACCAGAAGTGAATGAAAACAGTGATCGTTATTCATTGATATATGTTCCAAACGGCTTTTGCATTCCTGGTGGCAGGTTCCGAGAGCTGTACTATTGGGACACATACTGGATCATCAATGGACTCCTTTTGAGTGACATGAACGATACAGCCAAAGGAATCATTGAAAATCTACTAAGCCTTGTCCAAAAGATAGGTTTCATACCGAATGGCTCCAGAGTTTATTACTTGAACCGCTCACAACCTCCATTGCTAATCCAGATGATGAATAATTATTACAAAGCTACCAATGATTTTCAGTTCATCAAAAAAAATATCAAGACTTTGACAAAGGAGTTTGAATGGTGGCAGACGAACCGGAAAGTAAAATTTATCAAAGACAAAAAAACTTACAATATGTTCCGATATTATGCTCCCTCAAATGGACCAAGACCAGAATCTTACAGAGAGGATTATGAAATTGCTCAAACCCTTCCATCTGAAAGTGAGCGCACCCGATGGTATACTCGTATCAAGTCAGCAGCTGAAAGCGGGTGGGATTTCTCATCTAGATGGTTCATCAAAGACGGTGCAGGCAATGGCACACTTCTGGATGTTCACACGCCTAGTATAATACCTGTCGACTTAAATGCATTTCTCCACAAGAATGCTGTCTTACTGAGCGAATGGTGGTACATGATGGGCGATAAGTACCGAGGAAAGTACTTTAAGGAAATCGCAGAAAAACTTCTTGCTTCAATAAATGAGGTTTTATGGAATGAAAACATTGGATCCTGGTTTGACTATGACTTAATAAATAAACAGCACCGAAAATACTTTTTCCCTTCCAACATTGCCCCATTATGGACTGAGAGTTACTCTCAGCCGAAAAACTTCATGGCAGCCAAAGTCATTGAGTACATAAAACGGGAGAAAATCATCAAGGATGACTACACTGTTCACTATCATGGTATACCTTCTTCTTTGGAAAGGACAGGACAGCAATGGGACTTTCCCAACGCATGGGCCCCTGTTCAAGTGTTCTTGATTCAAGGCCTTGACCGCACCAATGTTCCCCAGGCGCAAGCAATCGCCTTGAAGCTAGCACAAGACTGGGTCCACTCCAACTACCTTGGTTTCCAAAAAACTGGTTTTATGTATGAAAAATACAATGTGGAAAAGGCAGGTGACAATGGTGGAGGTGGAGAGTATGAGTCTCAGATCAGTTTTGGATGGAGCAACGGAGTCGTTTTTGAAATGATGGATCGTTACGCCACAGGACTCTCCTCTGCAACCCTCACCAGATGATGGAGTGCTCTTCAGGCAGCCCTAAATGAGTGACTCAAGAAAGTCGTGGATAAGCAAATTGTGATATTTTCTAGCCTTTTAATGTTAAGTCTCTCCTTGACTGGGAAACAGAGTCAAGTAAAAAAAAAATTTAGCTCAGTTCCTACTTGAAGTACCTTGTTTTGCCGGACCGAAAGGTTTAGCCGATCTCTTTTAGTTAGTGTTTGCAACTTGAAGGTGTCAAAGATTTCTTCAATTCACACAATGTCTGCAAATATGATGAGGTAAAAGACTATGAAAGTTCAGAAAAAAACATTTTTTGACAGGACCTCTCCGCAGTATGTATGTATTCTCTGAATATCTTAGTTTCTTAAAAATTATATTTGTTTTTTCTGTTTGTTAAGTCAACAATGGCCATTTGTTACATAAGAACTCCAATTTCAACAAAATAGTACAATGAAATGCAAATTATTTCAGTAAAGACGTTTTAGATGAAAGTCTCTTTTGAGAGAAATATAAGTTCAAATGGGTTTTGCACAACTTTATCCTCTCCAGAATGCAAGACATTTGCGGACATTGGTTGCCTTAGGTAGATTTTTTGCTTTTGTTTGTAAAGTGTATAAAATAAATGTACATAAGTAAGTTTTTGTATCGCAGCTTGAGGCAATAATTTCAAAAAGGCCAAATTGAATTTAAATGCATGAACACCAGCAAATTAATAGGAGCATAGCTCAGCCATCAATTGATGTTTCTGCGATCAGTTAGAAGTACAGTCTTTATGACTTCTGCTGAATGTCAAGAGAGGAACTTGTTCACTTGAAACCAGACTTGATAATTTATTATTTATTCATTTGTCACGGCAGCTATGGCAAATTGTTGAAAACAATTTTGAATTTTAAAAAAAAATAATAAACACTTAAGTTCTTCTAGAAAAATTACTGAATTTGAACAAAAAATCACTTGTCTAGAGGTGCTTCTCTTCAAATGGTAAACCTCTAAATTCCAAAGTTTTAACTTTTGACTTTTTTAAAAATGTAGATTTTATCCGTGAAGTATTTTTGACAAGTTTTGCATTCAAGAAGGAATGCGTTTATAATCATTTCAATGTTGTACAGTTTAGTTCCTGTTTAGTCATATGTTTTTAATTTATTTTCAAGTCAAATTTTATATTGTTTGCATCTGTGTTTAAATTATGTTACTACTTAAACTGCAGAACCGTGCAAAGATTAAGGCAACAATTAGTTTCTTAAATTATTTGATGAGAGGGTTTGTAGCATGATATCTTGCAGTAGCAGGTACAACATTTTTATTTTTAAAACATAAAAGTTACCTATTTATTTTATCAATCCACTTGCAAGTGAAGTAATTCAAACAGGTAAAACTTATCAAAGAAAATGACAGATGATCATGGTAGACACTGAAAACCACCCTACTTTTTAAAGGAAATAAATTTAATTTTTTTAAATCTTCCATCAAAAAAAAGAAAAAAAAAAAAAAAAAAAAAAAGTACTCTGCGTTGTTACCACTGCTTAAGGGCGATCCCAATCACTAGTGAATTCGCGGCCGCCTGCAGGTCGACCATATGGGAGAGCTCC

the invention also provides a recombinant expression vector and a recombinant strain containing the coding gene BtTreh1 of the bemisia tabaci MED cryptic soluble trehalase.

The method for reducing the temperature tolerance of the bemisia tabaci comprises the step of feeding dsRNA of the gene BtTreh1 of the bemisia tabaci, wherein the dsRNA is obtained by amplifying the following primers:

BtTreh1-F：5’-TAATACGACTCACTATAGGGTCACCTGCCTTTTCCAC-3’，

BtTreh1-R：5’-TCCGATATTATGCTCCC-3’。

according to a specific embodiment of the invention, the agent for controlling bemisia tabaci comprises a dsRNA fragment of the bemisia tabaci MED cryptic soluble trehalase gene BtTreh 1.

The invention has the beneficial effects that:

according to the invention, cDNA of BtTreh1 gene is cloned from the hidden seed of Bemisia tabaci MED, and fluorescent quantitative PCR shows that the expression level of BtTreh1 gene is obviously reduced when the hidden seed of MED is stressed for 1h and 3h under the adverse temperature, and the expression level of the control is recovered after 5h of stress. The BtTreh1 gene dsRNA is fed, so that the high-temperature knockdown time of the bemisia tabaci MED cryptophyte adults is obviously reduced, and the low-temperature knockdown recovery time is increased.

The invention defines the expression time of the BtTreh1 gene in the hidden seeds of Bemisia tabaci MED and the key effect of the gene on the temperature tolerance of the hidden seeds of the MED, and provides a basis for a method for controlling the harm of Bemisia tabaci by temperature adaptability in the future.

Drawings

FIG. 1 shows the expression pattern analysis of BtTreh1 gene at gradient temperature in Bemisia tabaci MED cryptic;

FIG. 2 shows the changes of expression levels of three treatments of feeding BtTreh1 gene dsRNA, dsEGFP and feeding 10% sucrose solution;

FIG. 3 shows the high temperature knockdown time of Bemisia tabaci MED cryptomorphic adults after feeding BtTreh1 gene dsRNA, dsEGFP and feeding 10% sucrose solution for 3 h;

FIG. 4 shows the recovery time of tobacco whitefly MED cryptophyte adults after 3h feeding BtTreh1 gene dsRNA, dsEGFP and 10% sucrose solution.

Detailed Description