阅读说明：本技术 烟粉虱MED隐种染色质重塑因子Btbrm2及其编码基因和应用 (Bemisia tabaci MED cryptomorphic chromatin remodeling factor Btbrm2 and coding gene and application thereof ) 是由 吕志创 冀顺霞 王晓迪 申晓娜 刘万学 万方浩 于 2020-07-29 设计创作，主要内容包括：本发明涉及农业生物技术领域,具体地涉及烟粉虱MED隐种染色质重塑因子Btbrm2及其编码基因和应用。所述重塑因子的氨基酸序列如SEQ ID No：2所示。本发明明确了不同温度下Btbrm2基因在烟粉虱入侵种MED隐种与本地种Asia II 1隐种中表达模式的差异。降低该基因的表达直接降低了MED隐种对逆境温度的耐受能力。所获得的结果为进一步研究烟粉虱MED隐种的温度耐受性机理与表观遗传之染色质重塑的关系奠定基础,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 relates to the technical field of agricultural biology, in particular to a bemisia tabaci MED cryptic chromatin remodeling factor Btbrm2, and a coding gene and application thereof. The amino acid sequence of the remodeling factor is shown in SEQ ID No:2, respectively. The invention defines the difference of the expression modes of the Btbrm2 gene in the tobacco whitefly invasive species MED hidden species and the local species Asia II 1 hidden species at different temperatures. The reduction of the expression of the gene directly reduces the tolerance of the MED cryptic species to the adversity temperature. The obtained result lays a foundation for further researching the relationship between the temperature tolerance mechanism of the hidden species of the bemisia tabaci MED and the epigenetic chromatin remodeling, 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 chromatin remodeling factor is characterized in that an amino acid sequence is shown as SEQ ID No:2, respectively.

2. A diaphorina fumosorosea MED cryptic chromatin remodeling gene Btbrm2 encoding the diaphorina fumosorosea MED cryptic chromatin remodeling factor of claim 1.

3. The bemisia MED cryptic chromatin remodeling gene Btbrm2 of claim 2, wherein the nucleotide sequence is shown in SEQ ID No. 1.

4. A recombinant expression vector comprising the bemisia tabaci MED cryptic chromatin remodeling gene Btbrm2 of claim 2.

5. A recombinant strain comprising the bemisia tabaci MED cryptic chromatin remodeling gene Btbrm2 of claim 2.

6. The use of the bemisia tabaci MED cryptic chromatin remodeling gene Btbrm2 of claim 2 for controlling bemisia tabaci.

7. The use of the bemisia tabaci MED cryptic chromatin remodeling factor of claim 1 for controlling bemisia tabaci.

8. A method of reducing temperature tolerance of bemisia tabaci, comprising the step of feeding to bemisia tabaci the dsRNA of bemisia MED cryptic chromatin remodeling gene Btbrm2 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:

Btbrm2-F：5’-TAATACGACTCACTATAGGGTGTGATATGTCAGGGCT-3’，

Btbrm2-R：5’-TAATACGACTCACTATAGGGTACTCGGTGATTGGTGG-3’。

Technical Field

The invention relates to the technical field of agricultural biology, in particular to a bemisia tabaci MED cryptic chromatin remodeling factor Btbrm2, and a coding gene and application thereof.

Background

Bemisia tabaci (Gennadius)) belongs to the phylum Arthropoda, Insecta, Hemiptera, Bemisia, and is an important agricultural pest worldwide. The insect host has a wide range, can damage over 600 economic crops such as leguminous crops, solanaceae crops, compositae crops, cucurbitaceae crops, malvaceae crops and cruciferae crops in a mode of taking plant juice, secreting honeydew to induce sooty mould, spreading plant viruses and the like, and is a great pest which is necessary to deal with the crop production in China. Bemisia tabaci is a complex of species containing more than 36 cryptophytes. At present, the number of the existing aleyrodids hidden species in China is 15, including 2 invasive species and 13 local species. The hidden species of the invasive species MED are widely distributed in China. The local seeds are only distributed in local areas of China, for example, the AsiaII 1 hidden seeds are mainly distributed in local areas of the south and the middle, such as the areas of Hainan, Guangdong, Guangxi, Taiwan, Guizhou, Zhejiang and the like, and have no tendency of spreading to other areas.

With the change of global temperature, the successful adaptation of the hidden species of bemisia tabaci MED under different geographic environments has widely led people to the theoretical discussion of the invasion mechanism, and the molecular mechanism of the temperature adaptability is one of the research hotspots in recent years. In the experiment of heat shock selection of Bemisia tabaci, the survival rate of the invasive species Bemisia tabaci in the 2 generations is obviously improved, and the rapid improvement of the survival capability is an important strategy for the survival of the Bemisia tabaci in the severe environment. The mechanism of response of organisms to environmental variations within this short period of time is epigenetic related. Epigenetic processes increase the organism's response to the evolutionary potential for non-temperature stress and other environmental challenges, which are highly correlated with the background of global environmental warming.

Chromatin remodelling (chromatin remodelling) is one of the current research hotspots in the field of epigenetics, driving the replacement or rearrangement of nucleosomes, altering the spatial conformation of chromatin. ATP-dependent chromatin remodeling is an important epigenetic regulation mechanism for chromatin remodeling complex to utilize energy released by ATP hydrolysis to change chromatin structure, thereby regulating eukaryotic gene expression. Chromatin remodeling plays a key role in the expression regulation of genes related to biological defense, and the chromatin remodeling factor BRM can participate in the regulation of environmental stress on the biological adversity and plays an important role in regulating the expression of genes related to abiotic stress. Its role in temperature stress of bemisia tabaci is unknown.

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 available in the biological world for silencing certain genes in the insect body through RNAi technology

Therefore, some abilities of the insects are enhanced or lost, and the functional gene expression of the insects can be inhibited at a specific time, so that the development of the insects is stopped at a certain stage, and the aim of utilizing or preventing the damage of the insects is fulfilled. 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 bemisia tabaci MED cryptic chromatin remodeling factor.

Still another object of the present invention is to provide a gene encoding the above diaphorina fumonis MED cryptic chromatin remodeling factor.

Still another object of the present invention is to provide a recombinant expression vector containing a gene encoding the above bemisia tabaci MED cryptic chromatin remodeling factor.

Still another object of the present invention is to provide a recombinant strain containing a gene encoding the above bemisia tabaci MED cryptic chromatin remodeling factor.

The invention further aims to provide application of the coding gene of the bemisia tabaci MED cryptic chromatin remodeling factor.

According to the specific embodiment of the invention, the bemisia tabaci MED recessive chromatin remodeling gene Btbrm2 is cloned for the first time, and the full-length nucleotide sequence of cDNA is shown as SEQ ID No. 1:

ATGGCAGCTCCCACTCAAGACAATCTGAATGCTTTGCAGCACGCTATAGATTCAATGGAAGAGAAAGGTATGCAAGAGGATCCTAGATATTCTCAGCTCCTCGCTATCAGAGCTCGTCAAAATAACATGGAGCCCCCTCGACTTCCTGGATCGCAAGGATTTTGCGTATCCTCTGATAGCCCATCGAATTCCAAGCAAGTTTTGAGTCCCATCCAGTTGCAACAGTTACGAGTCCAAATCATGGCATATCGAGTTCTCGCTCGGAATCAGCCGTTGCCTCATCAACTTGCAGTAGCGGTTCAAGGGAAACGAATGGACTCGTCTCTCATTCAACCGGGTCTGGGACCTCCACCTCTCATGCCTCCAACCTCCGGTGGAAACACTCCAAGCCCACAAGCACGACCCACTGATTCAAGCAATGAAAACTCATCTGGAATGAGTGCAGCTCCTGTGTACCCTGTCGCACCCCCAACTTCACCCAACGTACCCCAAGGACCTCCGCCATCTCAGTTGCCCCCAGTAACCCCACCTCCCCCACAGTCATCCTCAACGATGGCTCCACCTCCTGCTGTTCCAGCACCACCTGCTCCATCTGTTCCTGCCCCTCTCCCACCCCCTGCTCTCAATTCAACACCCCCTGCGCCCCTCATGCCTCCCATTTCAGCACCGACACCACCTGTACCCTTGCAGCCACCAGTGACGTCACACGTCCATATCGCCCCACCCATGCCTCGGCTCCCCATCACGAGTCATCCCGTCATGGTTGTCCCTCCTCCTCCTAACTCAATGCTGAAACCGATGCCCAGTCCAGTTGTTCTGCCTCCTCCGCCACCACCACCCCCCAATCCGCAGCAACTTACGCAGCAACTTCAGTTACAAAAACAGAACAGAGTTACGACTATTCCTCGTCCAACTGGGATCGACCCTATACTAATTTTACAAGAAAGAGAAAATAGAGTCGCAGCTCGCATTGCCCATCATATGGAGAAGCTCTCCAATCTTCCGTCAACAGTGTCGGATGAAATTCGCCTCAAAGCTCAGATCGAACTTCGTGCTTTGAGAATCTTGAATTTTCAACGTCAGCTCCGAAGCGAGATCATATCTTGTGCTCGCAGAGATTCAACCCTGGAAACAGCCGTCAATATGAAAGCCTACAAGCGAGTCAAAACCCAAGGATTAAAAGAATCTCGCGCGACAGAAAAGTTGGAAAAGCAGCAGAAACTGGAAGCAGAGAGGAAACGGAGACAAAAACATCAAGAATATTTGGCAGCCATCATTCAGCACGGTAAAGACTTTAAAGAATACCATCGGAATAATCAAGCGAGGATCGTCCGGTTGAATAAAGCCATGCTTAGTTACCATGTGAATGCGGAGCGGGAGCAGAAGAAAGAACAGGAGAGGATCGAAAAGGAACGAATGAGACGCCTAATGGCAGAAGATGAAGAGGGTTACAGGAAACTCATCGATCAAAAGAAAGACAAGAGACTGGCATTCTTACTGTCTCAAACTGACGAATACATCACAAACTTGACAGAAATGGTCAAACAACACAAAGTTGAACAAAAACGTAAACTTGCAGAGAAAGCGAAAGAAGACAAGAGGAAAAAGAAAGAAACTGAGGATGCGGAGCGGAGAGCTTGCGTAATGGAAATGGAAAGTGGGAAAAAATTGAAGGGAGAAGAAGCTCCCTTGATGGTGGACCTAGGCACTTGGCTGGAAGCTCATCCTGGTTGGGAAGCATGTGAGGACTCAGAGGCAGAGACTGAAGAAGAGTCTGATTCTGATTCCGATCAAAATATCGAAGAAAAAATTGAAGAAGTCAAAGAAGAGAAGAAACCAAAAGAAGAACTTGATCCGAAAGAAGTTGTTGAAAAAGCGAAAAGTGAAGACGATGAATACAAAAATACCTCCGAAGAACTTACCTACTACAGTATTGCTCACACAATCAATGAAATTGTGACAGAACAAGCCTCTATCATGGTTAATGGTAAATTAAAAGAATATCAAATCAAGGGACTTGAGTGGCTAGTCTCTCTGTACAATAATAATCTCAATGGCATCCTGGCCGATGAAATGGGTCTTGGAAAAACCATCCAAACAATCGCATTGATCACATACTTGATGGAGATGAAAAAAGTAAACGGTCCATATTTAATCATCGTACCTCTTTCCACCCTTTCAAATTGGGTGTTGGAGTTTGAAAAATGGGCCCCATCTGTAAATGTTGTTGCGTACAAAGGCTCACCAGCTGTCAGACGTGCACTTCAAGCACAAATGAGATCATCTAAATTCAATGTGCTACTCACAACGTATGAATACGTTATCAAGGATAAAGCTGTTCTAGCCAAGTTGCACTGGAAGTACATGATCATCGATGAAGGGCATCGAATGAAGAATCACCATTGCAAATTGACACAAGTGCTAAATTCACATTATGTTGTTCCGCAAAGGCTCCTTTTGACTGGAACTCCGTTGCAAAATAAGTTGCCCGAACTGTGGGCTCTCCTCAATTTTCTTCTTCCGTCTATTTTCAAATCCTGCTCAACATTTGAACAGTGGTTCAATGCTCCTTTTGCCACAACTGGTGAAAAAGTGGAATTAAATGAGGAAGAAACTATTTTGATCATTCGCCGTCTACATAAAGTTTTACGTCCATTTTTGCTGCGACGTTTGAAGAAAGAAGTCGAGTCTCAACTTCCAGACAAAATCGAATATATCGTAAAATGTGATATGTCAGGGCTCCAGAGAGTCTTGTATAGGCACATGCAAAGTAAAGGGGTCCTTCTAACTGATGGCTCTGAAAAAGGTAACAAGGGCAAAGGTGGAGCCAAAGCACTGATGAATACAATTGTCCAACTTCGCAAGTTGTGCAATCATCCATTCCTGTTTCAACATATCGAAGAGAAGTTCTGCGATCATATCGGCTGTTCGTCAAATGGTGTTGTAAGTGGACCTGACCTTTATCGAGTCTCTGGTAAGTTTGAACTATTGGATCGAATTTTGCCAAAGTTGAAGGCCACCAATCACCGAGTACTTCTATTTTGTCAAATGACCCAGCTCATGAGCATAATGGAGGATTATTTCAACTGGCGAGGATTCTCGTACCTTCGTCTTGATGGAACAACCAAAGCGGAAGATCGAGGGGATTTATTGAAAAAATTTAACAGCGCCACCAGTGAATATTTCATATTCTTACTGAGCACACGAGCTGGTGGACTTGGCTTAAATCTTCAAGCTGCAGACACTGTAGTTATTTTCGACTCTGATTGGAATCCCCATCAGGATTTACAAGCGCAAGATCGAGCACATCGAATTGGTCAAAAGAACGAGGTACGAGTATTGAGGTTAATGACGGTCAATTCCGTTGAAGAACGAATCTTAGCAGCAGCTCGATACAAATTGAACATGGATGAAAAAGTCATCCAGGCAGGCATGTTCGATCAGAAGTCTACTGGAACTGAGAGGCAGCAGTTTTTGCATAGTATTTTGCATCAAGATGATGCAGAAGATGAGGAAGAGAATGAGGTTCCGGATGATGAAACTGTAAATCAGATGATTGCTCGCAGTGAAGCTGAATTTGACACTTTTCAAAAAATGGATGCTGAAAGACGCAAAGAAGAGTCTAAGGGCAAAAAATCCCGGCTTATTGAGGAGAGTGAACTACCAGACTGGTTGGTTAAAGATGACGCAGAGGTTGAAGCTTGGACGTACGAACAGGAAGAAGTGCAGATGGGACGAGGCTCAAGGACAAGAAAGGAAATTGACTACTCAGACAGCATGACAGAAAAGGAATGGTTAAAGGCCATTGATGATGGTATTGATGACTTTGATGAGATTGAAGATGAAGTGAAAGTGAAGAAAACTCGCAAACGGCGCAAAAAAGAAGAGGAGGAAGAGGAACCGGCCAAAAAACGAAGAAATGGTACAGAGAAGAATCCGCAAGAGAAAACACCACCTGCTAATTCAGGAGCGGATGCTCGAATGAAGAAGCAAATGCATAAATTGATGACGATTGTTGTAGAGTACACTGAACCTCAAGATTCGCGTGTTCTCAGTGAACCATTCATGAAACTACCATCTCGTCGTGAACTTCCTGATTATTATGAAGTCATCAAGAAACCCCTCGACATCAAGAAAATTCTCACAAAAATTGATGAAGGAAAGTACGAGGAACTGGATGATCTGGAGCGCGATTTCATGCAATTATGCAAGAATGCGCAGTTGTATAATGAAGAAGCATCACTCATCTATGAAGACTCAATTGTACTGCAGTCTGTTTTTACGAACGCTCGTCAGCGTTTGGAAAGTGAAGAGGAAGAAGCTCCGGAGGAAGAAGAAAAAGCGGCACCTGCTGAAGAGGAAGCCTCATCTGGAGCAGAATCGTCCTCTGTGAAAATGAAATTAAAATTGAAGGCATCAACCAAAGCGCCGAAAAATTCCGATGCCAAAACTGATACAAAGTCTACCCCACGATCAAGGAAAAGAACCTCGAAAAAATACATAAGTGACGATGAGAATGAAGATGATGCCGAATCAAGTAATGGCTAA

the amino acid sequence of the tobacco whitefly MED recessive chromatin remodeling gene Btbrm2 is shown in SEQ ID NO. 2:

MAAPTQDNLNALQHAIDSMEEKGMQEDPRYSQLLAIRARQNNMEPPRLPGSQGFCVSSDSPSNSKQVLSPIQLQQLRVQIMAYRVLARNQPLPHQLAVAVQGKRMDSSLIQPGLGPPPLMPPTSGGNTPSPQARPTDSSNENSSGMSAAPVYPVAPPTSPNVPQGPPPSQLPPVTPPPPQSSSTMAPPPAVPAPPAPSVPAPLPPPALNSTPPAPLMPPISAPTPPVPLQPPVTSHVHIAPPMPRLPITSHPVMVVPPPPNSMLKPMPSPVVLPPPPPPPPNPQQLTQQLQLQKQNRVTTIPRPTGIDPILILQERENRVAARIAHHMEKLSNLPSTVSDEIRLKAQIELRALRILNFQRQLRSEIISCARRDSTLETAVNMKAYKRVKTQGLKESRATEKLEKQQKLEAERKRRQKHQEYLAAIIQHGKDFKEYHRNNQARIVRLNKAMLSYHVNAEREQKKEQERIEKERMRRLMAEDEEGYRKLIDQKKDKRLAFLLSQTDEYITNLTEMVKQHKVEQKRKLAEKAKEDKRKKKETEDAERRACVMEMESGKKLKGEEAPLMVDLGTWLEAHPGWEACEDSEAETEEESDSDSDQNIEEKIEEVKEEKKPKEELDPKEVVEKAKSEDDEYKNTSEELTYYSIAHTINEIVTEQASIMVNGKLKEYQIKGLEWLVSLYNNNLNGILADEMGLGKTIQTIALITYLMEMKKVNGPYLIIVPLSTLSNWVLEFEKWAPSVNVVAYKGSPAVRRALQAQMRSSKFNVLLTTYEYVIKDKAVLAKLHWKYMIIDEGHRMKNHHCKLTQVLNSHYVVPQRLLLTGTPLQNKLPELWALLNFLLPSIFKSCSTFEQWFNAPFATTGEKVELNEEETILIIRRLHKVLRPFLLRRLKKEVESQLPDKIEYIVKCDMSGLQRVLYRHMQSKGVLLTDGSEKGNKGKGGAKALMNTIVQLRKLCNHPFLFQHIEEKFCDHIGCSSNGVVSGPDLYRVSGKFELLDRILPKLKATNHRVLLFCQMTQLMSIMEDYFNWRGFSYLRLDGTTKAEDRGDLLKKFNSATSEYFIFLLSTRAGGLGLNLQAADTVVIFDSDWNPHQDLQAQDRAHRIGQKNEVRVLRLMTVNSVEERILAAARYKLNMDEKVIQAGMFDQKSTGTERQQFLHSILHQDDAEDEEENEVPDDETVNQMIARSEAEFDTFQKMDAERRKEESKGKKSRLIEESELPDWLVKDDAEVEAWTYEQEEVQMGRGSRTRKEIDYSDSMTEKEWLKAIDDGIDDFDEIEDEVKVKKTRKRRKKEEEEEEPAKKRRNGTEKNPQEKTPPANSGADARMKKQMHKLMTIVVEYTEPQDSRVLSEPFMKLPSRRELPDYYEVIKKPLDIKKILTKIDEGKYEELDDLERDFMQLCKNAQLYNEEASLIYEDSIVLQSVFTNARQRLESEEEEAPEEEEKAAPAEEEASSGAESSSVKMKLKLKASTKAPKNSDAKTDTKSTPRSRKRTSKKYISDDENEDDAESSNG

the amino acid sequence has the typical structural characteristics of brm protein: HSA domain (406-478aa), BRK domain (541-585aa), ATPase domain (DEXDc:661-853aa and HELICC:1022-1106aa), SnAC domain (1201-1266aa), and Bromo domain (1325-1436 aa).

The expression mode of the Btbrm2 gene under temperature stress is analyzed, and the real-time fluorescent quantitative PCR result shows that the expression quantity of the Btbrm2 gene is obviously increased after the bemisia tabaci MED hidden species is subjected to low-temperature stress treatment for 5 hours; after the high-temperature stress treatment is carried out for 1h, the expression level of the Btbrm2 gene is obviously increased. After short-time high-temperature and low-temperature stress treatment, the expression level of the gene of the local Asia II 1 cryptophyte of the bemisia tabaci is obviously reduced. Comparing the expression level of Btbrm2 gene of different cryptophytes under the same temperature stress, the expression level of MED cryptophyte Btbrm2 gene after high and low temperature stress treatment is obviously higher than that of Asia II 1 cryptophyte.

The invention provides application of the bemisia tabaci MED cryptic chromatin remodeling gene Btbrm 2. RNAi is carried out on the cryptophyte of the bemisia tabaci MED, and the result shows that the high-temperature knockdown time of the cryptophyte MED imagoes fed with dsBtbrm2 is obviously reduced, and the low-temperature cold-induced stunning recovery time is obviously increased, which indicates that the Btbrm2 gene plays a key role in the temperature tolerance of the cryptophyte of the bemisia tabaci MED. The bemisia tabaci MED recessive chromatin remodeling gene Btbrm2 can be used for destroying temperature tolerance of bemisia tabaci, and further can be used for preventing and treating the bemisia tabaci.

The chromatin remodeling gene Btbrm2 is cloned from the aleyrodids MED hidden species for the first time, and the difference of the expression modes of the Btbrm2 gene in the aleyrodids MED hidden species and other hidden species under the stress of temperature is determined. In addition, the reduction of the expression of the gene directly reduces the tolerance of the MED cryptic species to high and low temperatures. The obtained result lays a foundation for further researching the relationship between the temperature tolerance mechanism of the hidden species of the bemisia tabaci MED and the epigenetic chromatin remodeling, 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.

Drawings

FIG. 1 conserved domain in the Bemisia tabaci MED cryptic Btbrm2 gene;

FIG. 2 shows the differences in expression patterns of the Btbrm2 gene in Bemisia tabaci MED cryptic species and AsiaII 1 cryptic species at different temperatures for treatments 1h (A), 3h (B), and 5h (C);

FIG. 3 shows the expression level changes of Btbrm2 under the conditions of feeding Btbrm2 gene dsRNA, feeding dsEGFP, feeding 10% sucrose solution and CK;

fig. 4 shows the effect of dsRNA treatment of Btbrm2 gene on whitefly MED cryptophyte adult heat resistance (a) and cold resistance (B): comparing the high-temperature knockdown time and the low-temperature cold-induced stunning recovery time of the Bemisia tabaci MED cryptomorphic adults fed with Btbrm2 gene dsRNA, dsEGFP and 10% sucrose solution under CK conditions.

Detailed Description