阅读说明：本技术 烟粉虱MED隐种多巴胺脱羧酶及其编码基因BtDDC和应用 (Bemisia tabaci MED cryptomorphic dopamine decarboxylase, coding gene BtDDC and application thereof ) 是由 吕志创 申晓娜 冀顺霞 梁林 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隐种多巴脱羧酶及其编码基因BtDDC和应用。烟粉虱MED隐种多巴胺脱羧酶的氨基酸序列如SEQ ID No.1所示。本发明明确了BtDDC基因在烟粉虱MED隐种中不同温度下的表达模式及该基因对MED隐种的温度耐受性所起的关键作用,可用于破坏烟粉虱温度耐受性,进而用于防治烟粉虱的危害及扩散。(The invention belongs to the technical field of agricultural biology, and particularly relates to bemisia tabaci MED cryptomorphic dopa decarboxylase, a coding gene BtDDC and application thereof. The amino acid sequence of the Bemisia tabaci MED cryptomorphic dopamine decarboxylase is shown as SEQ ID No. 1. The invention defines the expression modes of the BtDDC gene in different temperatures of the Bemisia tabaci MED cryptic species and the key effect of the gene on the temperature tolerance of the MED cryptic species, and can be used for destroying the temperature tolerance of the Bemisia tabaci and further preventing and controlling the harm and the diffusion of the Bemisia tabaci.)

1. The Bemisia tabaci MED cryptomorphic dopamine decarboxylase is characterized in that the amino acid sequence of the Bemisia tabaci MED cryptomorphic dopamine decarboxylase is shown as SEQ ID No. 1.

2. A Bemisia tabaci MED cryptogenic dopamine decarboxylase gene BtDDC, characterized in that it encodes the Bemisia tabaci MED cryptogenic dopamine decarboxylase of claim 1.

3. The bemisia MED cryptotrophic dopamine decarboxylase gene BtDDC according to claim 2, characterized in that its nucleotide sequence is as shown in SEQ ID No.2 or SEQ ID No. 3.

4. A recombinant expression vector comprising the bemisia tabaci MED cryptogenic dopamine decarboxylase gene BtDDC of claim 2.

5. A recombinant strain comprising the bemisia tabaci MED cryptotrophic dopamine decarboxylase gene BtDDC of claim 2.

6. The use of bemisia tabaci MED cryptomorphic dopamine decarboxylase gene BtDDC according to claim 2.

7. The use of the bemisia tabaci MED cryptotrophic dopamine decarboxylase gene BtDDC of claim 2 for reducing bemisia tabaci MED temperature tolerance.

8. A method of reducing the temperature tolerance of the bemisia tabaci cryptic MED, comprising the step of feeding the bemisia tabaci MED, the dsRNA of the bemisia tabaci MED cryptic dopamine decarboxylase gene BtDDC of claim 2.

9. The method of reducing the temperature tolerance of the bemisia tabaci cryptic MED of claim 8, wherein the dsRNA is amplified using the following primers:

BtDDC-F：5’-TAATACGACTCACTATAGGGCGCAGAACAAACCACAAT-3’；

BtDDC-R：5’-GCCAAAACCAGAGCAATC-3’。

10. an agent for controlling bemisia tabaci, comprising the dsRNA fragment of bemisia tabaci MED cryptogenic dopamine decarboxylase gene BtDDC of claim 2.

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to bemisia tabaci MED cryptomorphic dopa decarboxylase, a coding gene BtDDC 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 RNA interference (RNAi) phenomenon 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 Bemisia tabaci MED cryptomorphic dopamine decarboxylase.

The invention further aims to provide the coding gene BtDDC of the bemisia tabaci MED cryptomorphic dopamine decarboxylase.

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.

It is a further object of the present invention to provide a method for reducing temperature tolerance of aleyrodids cryptica MED.

According to the specific embodiment of the invention, the amino acid sequence of the Bemisia tabaci MED cryptomorphic dopamine decarboxylase is shown as SEQ ID No. 1:

MEGTVFTPNMEVAEFQDFAKAMIDYISKYMSSIRDRPVLPKLEPGYLRPLIPESAPEEPESWHNVMEDIERVIMPGVTHWHSPRFHAYFPTACSYPAIVADMLSDAIACIGFTWIASPACTELEVVMMDWLGKMLNLPDDFLACGKKGGGGVIQGTASEATLVALLGAKTRAIRRTKEEHPDWTDLEIASKLVAYCSKQAHSSVERAGLLGSVPFRLLPTDDMYRLQGNTLEEAIKKDKADGLVPFYVVATLGTTSCCSFDLLKEIGPVCKNEEVWLHVDAAYAGSAFICPEYQYLLEGVEYAESFNFNPHKWMLINFDCSAMWLKNPDEVVNAFNVDPLYLKHDHQGAAPDYRHWQIPLGRRFRSLKLWFVLRLYGIKNLQTHIRHQIGLAHQFEAYVNEDEEFELFNEVLMGLVCFRVKGSNELNEQVLNRINKKGKIHMVPSKIKDVFFLRFAVCSRFTNAEDVKYSWSEVKATTQEIKKELAKQ

the bemisia tabaci MED cryptomorphic dopamine decarboxylase provided by the invention has the typical structural characteristics of DDC protein: comprises a conserved domain of pyrrole-dependent decarboxylase, which is positioned at 44-421 of the sequence, wherein a site for connecting the phenylpyridine is contained, and is positioned at 305-326.

According to the specific embodiment of the invention, the genome nucleotide sequence of the coding gene BtDDC of the tobacco whitefly MED cryptomorphic dopamine decarboxylase is shown as SEQ ID NO. 2:

TACTATAGGGCGAATTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCGGCCGCGGGAATTCGATTGGATCGCCCTTCTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCGTCGTTTTATTTTTTCGTCCACCACCCATTCACCATGGAGGGCACCGTTTTTACCCCAAACATGGAGGTCGCGGAGTTCCAAGACTTTGCAAAGGCGATGATCGATTATATTAGCAAATACATGTCATCAATTCGAGACAGGCCTGTTCTACCTAAATTGGAGCCAGGATACTTGAGACCTTTGATCCCGGAGTCTGCACCAGAGGAACCAGAATCATGGCATAATGTTATGGAAGATATTGAAAGAGTTATTATGCCAGGAGTAACTCACTGGCATTCACCCCGCTTCCATGCATACTTCCCGACAGCCTGCTCTTACCCTGCCATAGTTGCTGACATGTTAAGTGACGCCATTGCTTGCATTGGATTTACCTGGATCGCAAGTCCAGCTTGCACAGAACTCGAGGTTGTGATGATGGACTGGCTCGGTAAGATGCTTAATCTCCCTGATGACTTTTTGGCCTGTGGAAAGAAAGGAGGTGGTGGTGTTATTCAGGGCACTGCTAGTGAGGCAACTCTTGTCGCCCTTCTGGGTGCCAAAACCAGAGCAATCCGCCGAACAAAAGAAGAGCATCCTGATTGGACAGATCTTGAAATCGCCTCCAAATTGGTAGCGTACTGCTCCAAACAAGCCCACTCCTCGGTGGAACGAGCAGGTCTTCTGGGAAGCGTCCCGTTCAGACTCCTACCTACAGACGATATGTACAGATTACAAGGCAACACTTTAGAAGAAGCTATCAAAAAAGACAAAGCTGACGGTCTCGTTCCTTTCTATGTTGTTGCAACTCTAGGAACGACCTCCTGCTGTTCATTTGATCTGCTCAAAGAAATTGGACCAGTGTGTAAAAATGAAGAAGTTTGGCTTCATGTTGATGCTGCGTATGCAGGATCTGCCTTCATTTGTCCCGAGTATCAGTATTTGTTAGAAGGGGTAGAGTACGCTGAGTCATTCAATTTTAATCCTCATAAGTGGATGCTCATAAACTTTGACTGCTCAGCCATGTGGTTAAAAAATCCAGATGAAGTTGTAAATGCATTCAATGTTGATCCTCTTTACCTGAAGCACGACCACCAAGGAGCTGCTCCTGATTATAGGCACTGGCAAATTCCACTCGGCAGGCGATTCCGTTCATTGAAATTGTGGTTTGTTCTGCGATTGTATGGAATAAAGAACCTTCAAACCCACATTCGTCATCAAATTGGACTCGCCCATCAGTTTGAAGCGTACGTCAATGAAGATGAGGAGTTTGAATTATTCAACGAAGTACTCATGGGTCTTGTCTGCTTCAGAGTTAAGGGCTCCAATGAATTGAACGAACAAGTGCTGAACAGGATCAACAAAAAAGGCAAAATCCACATGGTTCCCTCAAAAATCAAGGATGTCTTCTTCCTCCGTTTTGCCGTCTGTTCACGTTTCACAAACGCCGAAGATGTCAAATACTCGTGGAGTGAAGTCAAAGCCACAACCCAAGAAATCAAAAAAGAGCTAGCCAAGCAGTGATTGAAAGTTCCGACAAATATTTAAGTTATTCCACCTCCATGTATTATCTTTGTTAAGTTTTAGTACATTTAATCTCAAGATTCAATGGAAAAGCTCTCTATTTTTAGTAAGTTATTTTTACAGATTGACTATTCAAGCAGCACTAATGTTTTGTGCTCCAGGAGCCAAGCTCAAAGTTTTTAGGAACTCATTGAGTCTCACCCTATCTTGAATAACGAAATGAAAAAACTTAGCATGAAGATGAAATGGATTAAATTAAAATTTCTTCTAAGTGTAATTGAAGAATTGCGTTAAATATATATGCTTTTATTCCTCTCACTGCGAAAAGTATCAAAGACGATTCTCTAATAGCATAACTTCATAATTTCATTATTTAAAAATATTTTATACTTCAACCTCCGAACACCAAAGGGCACAGCCATGTCAACAAATCAACAACTACCAGACGGAGGAATGTATCTTAATTGCACTGTTTCAGTATGCTTGCTAATTTTTTTTCTTTCAAGGAAAACTGTTTTATGAATTTTCTTGAAAACTCTAATGATTTTTCTTTACCTTCGTGAGAAAAGACTCTAAAATTTTCAGATTGATTCGTGCGATGGTTCTTGCATGAAAATATAAAATCTTTCTAAAAACATTGAAACAGTCCAGGCAAGATATGTTCCTTTGTGCAGGAGACGGCAAAATGTCTCATTTCCTAAAAATTCGTTTGAGTGTCTGAAGAATTTGATGGATATTTCATCGTAGGCACTAAATAAAAGTGTAGCATGGCTGTTGGCATCTTCCTTTGATCAAGTGAAATTGTATCCAATAGTTTTGATTGTTAAGTTTGTTGTTTTCAATGCATTGTTCCAGTATTAGCAGAAAATTTATTATTTTACGATTACAATTATTGTTATTTATGGATGTAATTATAATTGTATGTGTAAGTGTAGATTTTAACTTTTTATGACAGGAATTGAATTTTAGACCATTCGTTTTTCAAGGGCCCTCATCCGATGCCCATTTCCTTGATTCTAGACAATTTTAATCAAATCATGATCTGTGAAAAACAAGTTTTATGATGCTTGCCAATGTGGTTTCTTTTTTTTTCTTTTTTTAAACTTATCTCACTCCGTACTTTGTGAATGATGAGCTGTGACATTATATGCTAGTATTTCAAAAATTTAATAATTTTTTGTAAAATTAAGTGTTTCTTATTTTATAGTCTTTTCATCCCTCGAAACACCATCTGTGACACCATTAATTCTTATTCTAATACACCTTAAGATCATCTCCAATTTAATGAAGATTTCATACATTTTGTTTTTTTTATAATATGAAAATTACTAATGTAAAATTTTCTTATCTTTTTGAATTGACTCATTTGCTCAAGCTACTTTTCAAAATAAAAATTCCAATTATTCTGTCAGCCTCAAAAAAAAAAAAAAATCTCAGTCGCTGAGTAGCACATAAATATTTAAAATCCTTGATTGATTGCCTAAAATTTCTAAGCTCATTTCAAAATCTGTATGTTAAATAGAATTCTATAGTCATTTGTATCATTTTGAATTGTCAAATTTTAAAAATTTCAGTCTCCTCACCTGTGTTGTCGGCTATTCTAATTCCCTCCTTAAATGTTCATCATCGGTGTTTAAAGAGCCTCAGGCAAGATTTTATGTTTGAATTGAATTGTTTTTTCAACATTCTACAATTGATATCTAGAGACAATGCATTAAAAGTTTAATGTTTTGTATGAGAAACTTTGTTCAATATTGCGCTAAAGTATTTTAGAAAAGTCAAATACCACACACCCAATCCCCCCCATTTGGATTAAATTATTGCAATCTTGTTCTAGTCTGAAAAGCAGCACAGTTTGAAGGTTTCACCAATGATCGTAAACATAAAAATCCATTTAATTAAAGAATTAACATAGAAAGGTTAGATAAGACTCTTGTAATAAAAGACACAAGACAATATAATGAATAATAATTTATTGTAAAAAGTTTAATTTATTC

the cDNA sequence of the coding gene BtDDC of the Bemisia tabaci MED cryptomorph dopamine decarboxylase is shown in SEQ ID NO. 3:

ATGGAGGGCACCGTTTTTACCCCAAACATGGAGGTCGCGGAGTTCCAAGACTTTGCAAAGGCGATGATCGATTATATTAGCAAATACATGTCATCAATTCGAGACAGGCCTGTTCTACCTAAATTGGAGCCAGGATACTTGAGACCTTTGATCCCGGAGTCTGCACCAGAGGAACCAGAATCATGGCATAATGTTATGGAAGATATTGAAAGAGTTATTATGCCAGGAGTAACTCACTGGCATTCACCCCGCTTCCATGCATACTTCCCGACAGCCTGCTCTTACCCTGCCATAGTTGCTGACATGTTAAGTGACGCCATTGCTTGCATTGGATTTACCTGGATCGCAAGTCCAGCTTGCACAGAACTCGAGGTTGTGATGATGGACTGGCTCGGTAAGATGCTTAATCTCCCTGATGACTTTTTGGCCTGTGGAAAGAAAGGAGGTGGTGGTGTTATTCAGGGCACTGCTAGTGAGGCAACTCTTGTCGCCCTTCTGGGTGCCAAAACCAGAGCAATCCGCCGAACAAAAGAAGAGCATCCTGATTGGACAGATCTTGAAATCGCCTCCAAATTGGTAGCGTACTGCTCCAAACAAGCCCACTCCTCGGTGGAACGAGCAGGTCTTCTGGGAAGCGTCCCGTTCAGACTCCTACCTACAGACGATATGTACAGATTACAAGGCAACACTTTAGAAGAAGCTATCAAAAAAGACAAAGCTGACGGTCTCGTTCCTTTCTATGTTGTTGCAACTCTAGGAACGACCTCCTGCTGTTCATTTGATCTGCTCAAAGAAATTGGACCAGTGTGTAAAAATGAAGAAGTTTGGCTTCATGTTGATGCTGCGTATGCAGGATCTGCCTTCATTTGTCCCGAGTATCAGTATTTGTTAGAAGGGGTAGAGTACGCTGAGTCATTCAATTTTAATCCTCATAAGTGGATGCTCATAAACTTTGACTGCTCAGCCATGTGGTTAAAAAATCCAGATGAAGTTGTAAATGCATTCAATGTTGATCCTCTTTACCTGAAGCACGACCACCAAGGAGCTGCTCCTGATTATAGGCACTGGCAAATTCCACTCGGCAGGCGATTCCGTTCATTGAAATTGTGGTTTGTTCTGCGATTGTATGGAATAAAGAACCTTCAAACCCACATTCGTCATCAAATTGGACTCGCCCATCAGTTTGAAGCGTACGTCAATGAAGATGAGGAGTTTGAATTATTCAACGAAGTACTCATGGGTCTTGTCTGCTTCAGAGTTAAGGGCTCCAATGAATTGAACGAACAAGTGCTGAACAGGATCAACAAAAAAGGCAAAATCCACATGGTTCCCTCAAAAATCAAGGATGTCTTCTTCCTCCGTTTTGCCGTCTGTTCACGTTTCACAAACGCCGAAGATGTCAAATACTCGTGGAGTGAAGTCAAAGCCACAACCCAAGAAATCAAAAAAGAGCTAGCCAAGCAGTGA

the invention also provides a recombinant expression vector and a recombinant strain containing the bemisia tabaci MED cryptic dopamine decarboxylase gene BtDDC.

The method for reducing the temperature tolerance of the cryptophyte MED of the bemisia tabaci comprises the step of feeding dsRNA of the cryptophyte dopamine decarboxylase gene BtDDC of the bemisia tabaci, wherein the primer sequence for amplifying the dsRNA is as follows:

BtDDC-F：5’-TAATACGACTCACTATAGGGCGCAGAACAAACCACAAT-3’；

BtDDC-R：5’-GCCAAAACCAGAGCAATC-3’。

the invention provides a pesticide for controlling bemisia tabaci, which comprises a dsRNA segment of a bemisia tabaci MED cryptic dopamine decarboxylase gene BtDDC.

The invention has the beneficial effects that:

the cDNA of the BtDDC gene is cloned from the hidden seed of Bemisia tabaci MED, and the fluorescent quantitative PCR shows that the expression quantity of the BtDDC gene is obviously increased within 5 hours of high-temperature and low-temperature stress; the BtDDC gene dsRNA is fed to discover 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 modes of the BtDDC gene in different temperatures of the hidden seeds of Bemisia tabaci MED and the key effect of the gene on the temperature tolerance of the hidden seeds of MED, and the BtDDC gene can be used for destroying the temperature tolerance of Bemisia tabaci and further preventing and controlling the harm and the diffusion of the Bemisia tabaci.

Drawings

FIG. 1 shows the results of analysis of expression patterns of BtDDC gene at gradient temperature in Bemisia tabaci MED cryptic species;

FIG. 2 shows the change of expression levels of BtDDC genes in three treatments of feeding BtDDC gene dsRNA, dsEGFP and feeding 10% sucrose solution;

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

FIG. 4 shows the recovery time of Bemisia tabaci MED Cryptodermia at low temperature after feeding BtDDC gene dsRNA, dsEGFP and feeding 10% sucrose solution for 3 h.

Detailed Description