CN107630026B

CN107630026B - Extremely dry-tolerant erythroaldehyde dehydrogenase gene and encoded protein thereof

Info

Publication number: CN107630026B
Application number: CN201711076388.3A
Authority: CN
Inventors: 张道远; 杨红兰; 王建成; 李小双
Original assignee: Xinjiang Institute of Ecology and Geography of CAS
Current assignee: Xinjiang Institute of Ecology and Geography of CAS
Priority date: 2017-11-06
Filing date: 2017-11-06
Publication date: 2020-10-30
Anticipated expiration: 2037-11-06
Also published as: CN107630026A

Abstract

The invention discloses an extremely dry-tolerant plant erythroaldehyde dehydrogenase gene and a protein coded by the geneScALDH2B2，ScALDH10A1AndScALDH11A1the cDNA sequence of SEQ ID NO:1-3, and the amino acid sequence encoded by the amino acid sequence is SEQ ID NO: 4-6; the lengths of the coding regions of the genes are respectivelyScALDH2B2The gene is 1584bp in length,ScALDH10A1the gene is 1521bp and has high selectivity,ScALDH11A1is 1491 bp; the ScALDH genes are all abscisic acid-dependent and respond to three abiotic stresses of drought, high salt and low temperature, but the response modes are different. The function verification is carried out by transforming prokaryotic cells and arabidopsis thaliana, and the result shows that: compared with wild type, the ScALDH2B2, ScALDH10A1 and ScALDH11A gene lines have the characteristics of drought resistance and salt tolerance enhancement. The invention provides important gene resources for cultivating new varieties of crops with abiotic stress resistance by applying a genetic engineering technology.

Description

Extremely dry-tolerant erythroaldehyde dehydrogenase gene and encoded protein thereof

Technical Field

The invention relates to an extremely dry-tolerant erythroaldehyde dehydrogenase gene and a protein coded by the same, and belongs to the technical field of molecular biology.

Background

The shortage of water resources and the salinization of soil are global problems restricting agricultural production at present, and how to promote the sustainable development of agriculture becomes one of the key problems which need to be solved urgently by modern plant breeding work, so that the improvement of the stress resistance of plants is always the goal pursued by phytologists. The identification and research of plant stress resistance genes are basic researches for disclosing plant stress resistance mechanisms on a molecular level. The stress resistance of plants belongs to a quantitative trait, involving the action of a large number of genes (Cushman and Bohnert 2000). The traditional breeding period is long; certain excellent traits are difficult to inherit, and the difficulty is brought to the improvement of single traits of plants. Plants produce excessive amounts of ROS under various stress conditions of high salinity, drought, cold damage, heavy metals, mechanical damage, nutrient deficiency, pathogens, etc., causing toxicity to plant cells (Bartels and Sunkar 2005). Many organisms acquire some characters suitable for extreme environmental conditions in the long-term evolution process, and can effectively eliminate or resist ROS poison. The enhancement of aldehyde dehydrogenase activity is just an important way to scavenge ROS (Rodrigues et al 2006). A plurality of aldehyde dehydrogenase genes which resist the up-regulated expression of salt damage, water deficiency and other adversities are identified in plants such as arabidopsis thaliana, grapes and the like, and the aldehyde dehydrogenase genes are proved to have important functions in resisting adversity stress of the plants. Therefore, by using genetic engineering means, genes controlling these traits can be introduced into plants, thereby improving their stress resistance and achieving the desired goal that is difficult to achieve by conventional breeding.

The continuous production of Reactive Oxygen Species (ROS) in plants under stress conditions leads to increased oxidative stress and cell damage. This process produces over 200 aldehydes, the excess of which causes metabolic disturbances in the plant body and lipid peroxidation, and ALDH is the "aldehyde scavenger" in the lipid peroxidation process (Wei et al, 2009; Surendra et al, 2013). ALDH is an important functional protein, participates in different important physiological processes such as plant growth and development, various defense reactions and the like, and plays an important role in drought resistance and salt tolerance (Kotchoni and D.Bartels.2003). However, only a few plant ALDH family members have been studied so far (Chad et al.2013).

Although the ALDH gene has been cloned from different plant genomes, it is less studied in terms of protein structure and enzyme activity; the physiological function research on the ALDH gene family of plants mainly focuses on the expression pattern of the ALDH gene family in monocotyledonous and dicotyledonous plants. The physiological function and biological significance of ALDH genes in many plants remains unclear. The erythrosine is widely distributed in Guerbantong Gute desert, can adapt to rapid change of water in the desert and is a dominant species in desert biological skinning (Zhang et al, 2007). Under an extremely dry state, the erythrodons dentata is covered on the surface of the desert like a layer of gray black 'shell' and is in a dormant state; upon encountering precipitation, the erythrosine "recovers" to a fresh green color and restores photosynthesis rapidly through the rehydration process. The research laboratory researches the distribution (Zhang et al,2007), ecological effect (Zhang et al, 2007; Zhang et al,2009) of the species, the form (Weimei and the like, 2009) in the rehydration process, the ultrastructure (Weimei and the like, 2009; Zheng et al,2011), the photosynthetic physiology (Li et al, 2010; Zheng et al,2011) and the like, and clearly shows the form structure and physiological and biochemical basis of the dehydration stress resistance of the species. The subject group further performs preliminary excavation on the crenella denticulata stress-resistant gene by analyzing the crenella denticulata transcriptome. Through analyzing the gene structure, the drought response mode and the gene stress resistance under different stress conditions (salt, drought and PEG), the ScALDH21 has excellent drought resistance and salt tolerance, the stress resistance mechanism of the erythropteris denticulata is similar to that of higher plants, and the aldehyde dehydrogenase (ALDH) family is predicted to have excellent molecular stress resistance breeding gene resources.

Therefore, the analysis of the stress resistance function of the ScALDHs family gene has important significance for the research of the stress resistance mechanism of the erythrodentis and the directional culture of new plant varieties.

Disclosure of Invention

The first purpose of the invention is to provide 3 aldehyde dehydrogenase genes from extreme dry-resistant erythrosine aiming at the limitation of the development and research of the desert stress-resistant plant erythrosine stress-resistant gene, which are named as ScALDH2B2, ScALDH10A1 and ScALDH11A1 respectively, and the nucleotide sequences of the genes are shown as SEQ ID NO. 1-3.

The second purpose of the invention is to provide aldehyde dehydrogenase proteins ScALDH2B2, ScALDH10A1 and ScALDH11A1 coded by 3 ScALDH genes of an extremely dry-tolerant Erythrophlia denticulata plant, wherein the amino acid sequences of the proteins are shown in SEQ ID NO: 4-6.

The third purpose of the invention is to apply the 3 odontoid erythropteris ScALDH2B2, ScALDH10A1 and ScALDH11A1 genes in breeding stress-resistant organisms, especially in abiotic stress-resistant organism varieties.

The extremely dry-tolerant erythroaldehyde dehydrogenase gene is named as ScALDH2B2, ScALDH10A1 and ScALDH11A1 respectively;

the length of a coding region of the aldehyde dehydrogenase gene ScALDH2B2 is 1584bp, the nucleotide sequence of the coding region SEQ ID NO.1 codes 527 amino acids, and the amino acid sequence SEQ ID NO. 4;

the length of the coding region of the aldehyde dehydrogenase gene ScALDH10A1 is 1521bp, the nucleotide sequence thereof is SEQ ID NO.2, the coding region codes 506 amino acids, and the amino acid sequence thereof is SEQ ID NO. 5;

the length of the coding region of the aldehyde dehydrogenase gene ScALDH11A1 is 1491bp, the nucleotide sequence thereof is SEQ ID NO.3, 496 amino acids are coded, and the amino acid sequence thereof is SEQ ID NO. 6.

The nucleotide sequences SEQ ID NO.1-3 are respectively subjected to prokaryotic expression vector construction according to a conventional method, and are transduced to prokaryotic cells DE3 to respectively obtain three prokaryotic transformants which are transformed with ScALDH2B2, ScALDH10A1 and ScALDH11A1 and have the stress resistance function.

The nucleotide sequences SEQ ID NO.1-3 are respectively constructed with plant expression vectors according to a conventional method and transformed into Arabidopsis plants to respectively obtain three plant transformants which are transformed with ScALDH2B2, ScALDH10A1 and ScALDH11A1 and have stress resistance.

The recombinant proteins ScALDH2B2, ScALDH10A1 and ScALDH11A1, ScALDH2B2 and ScALDH11A1, were used as enzymes of the aliphatic aldehyde dehydrogenase/reductase family, and ScALDH10A1 was used as an aromatic aldehyde dehydrogenase/reductase.

The application of the extremely dry-tolerant erythroaldehyde dehydrogenase gene in the preparation of plant stress-resistant breeding and improvement.

The extreme dry-tolerant erythroaldehyde dehydrogenase gene and the coding protein thereof are respectively named as ScALDH2B2, ScALDH10A1 and ScALDH11A1 genes, the lengths of coding regions are 1584bp, 1521bp and 1491bp respectively, and the nucleotide sequence of the characteristic sequence of the coding region shown as SEQ ID NO.1-3 is as follows:

SEQ ID NO.1 ScALDH2B2 nucleotide sequence

ATGGCGATGAGGGCGATGAGCAGGAAGCTCCTGTCGCGGGCGCTGCGACCTGCAGCGGTTAGTCGAAGCTACGGCGCGGCAGCGGCAGCCGCGGAGGAGCTGGGGAGCCCCATGAAGGCGCCAGTGGACGTGAAGCTCACCCAGCTGCTGATCGACGGGGAGTTCGTGGACGCCGCGTCGGGGAAGACGTTCGGGGTCATCGACCCCAGAAGCGAGCAAGTCATCGCGGAGGTCGCCGAGGGCGACGCGGAGGACGTCAACCGCGCGGTGCGCGCTGCAAGGAAGGCGTTCGACCATGGGCCGTGGCCGAAAATGCCCGCCCACAAGCGCGGCGAGATTCTCCTCAGGTATGCGGACTTGTTGGACCAGCACGCGGATGAGCTCGCGGCGCTGGAGACGATGGACTCTGGGAAGCCGTACGAGCAAGCGCGCTACGGGGAGGTGCCGATGGTGTCGCGGCAGTTCCGATACTACGCGGGGTGGGCGGACAAGATATACGGCACGACGGGGCCGTCTGATGGAAGCCACGCGGTGCACACGCTCCACGAGCCGTTGGGTGTGGTGGGGCAGATCATCCCCTGGAACTTCCCCATCGTCATGTACTGCTGGAAGGTGGCGCCTGCGCTGGCAACGGGGAACTGCGTCGTGCTAAAGACCGCGGAGCAGACGCCGCTGTCCGCCCTTCTGGCCGGGAAGCTGGCGTTGGAGGCCGGCATTCCCCCGGGCGTGCTCAACATCGTCTCCGGGTACGGCCCCACCGCGGGCGCCGCCATAGCCGAGCACATGGACGTCGACAAGGTCGCCTTCACGGGCTCCACCGAGGTCGGGAAGCTCGTCATGGGGGCGGCAGCGCGCAGCAACTTGAAGCCGGTGACGCTGGAGCTCGGAGGGAAGTCGCCCATGATCGTGTGCGAGGACGCGGACGTGGACGCTGCCGTGGAGCTGGCGCACTTCGCCATCTTCTTCAACCAGGGGCAGTGCTGCTGCGCGGGGTCGCGCACGTTTGTGCACGAGAGCATCTACGACGAGTTCGTGGAGAAGTCCAAGGCGCGCGCCCTGAAGCGCGTTGTGGGCGACCCTTTCAGGAGCGGCGTGGAGCAGGGCCCTCAGGTCAACAAGGAGCAGTTTAACAAGGTCCTCTCGTACATCGAGAGCGGGCAGCAGCAGGGCGCCAACCTCCTCACAGGTGGAGGTCGCTTGGGAAACGTAGGCTACTACATCAAGCCCACAATCTTCACTGACGTGAAAGACGGTATGAAGATCTTCGACGAGGAGATCTTCGGTCCTGTGCAGACGATTGCCAAGTTCAAAACCTTGGAAGATGTGGTGCAGCGGGCCAACAACACAGTGTACGGATTGGCAGCGGGCATTTTCAGCAACAACTTGAACACGGTGAACACGTTGAGCCGGGCACTGCGAGCGGGCACCGTGTGGGTGAACTGCTTCGACGTGTTCGACGCCACCATTCCGTTCGGGGGGTACAAGCAGAGCGGCATCGGGCGGGAGAAGGGCAAGGAGGCCCTGGACAACTACACGCAGGTGAAGGCGGTGGTGACGCCTATCCACAACCCCGCGTGGCTGTAA

SEQ ID NO.2 ScALDH10A1 nucleotide sequence

ATGGGTCTTCACGCTGAGATTGTGCCCCAGCGCCGCCTCTTCATCGACGGCGACTGGGTGCAGCCGCGCCAGGGCAAGCGCATTCCCATTGTCAATCCCACCACGGAGGAGAGCGTGGGGGACATTCCAGCTGCAACCTCGGAAGACGTCTATGCTGCAGTGAAGGCGGCTAAGGAAGCGTTACATCGCAACAATGGCAAGGACTGGTCTAAGGCCACTGGAAAGCACCGCGCAACCTACCTCCGAGCCATTGCCAAGAGGGTGGCTGAGAGAAAAGACGAGCTTGCGAAGCTGGAGTCCATCGACTGTGGCAAGCCTCTCGATGAAGCGGCGTGGGATATGGATGATGTATCAGGGTGTTTTGAGTACTACGCGGACCTGGCAGAGAAGTTGGATGAGAGGCAGTATGCTCCTCTGGAACTTCCCATGGAGCAATTCAAATGCAACATATTGCGACAGCCTGTTGGAGTTGTTGCTCTTATCACACCCTGGAATTACCCACTTTTGATGGCTACTTGGAAAGTTGCTCCAGCGCTTGCAGCTGGATGCACTGCCATTCTGAAACCATCTGAGATTGCCTCTGTCACTTGCTTGGAGTTGGCGAGTATTGCCAAAGAAGTTGGACTTCCGAACGGAGTCTTGAATGTGATCACTGGATATGGACAGGAAGCTGGTGCACCCTTGGCCTCACATCAGGATGTAAACAAGGTAGCTTTTACTGGAAGCACAGATACTGGCAGGTCTATCATGTCCGCGGCATCACAGCTCATCAAGCCAGTAACATTGGAATTGGGTGGAAAGAGCCCCATCATAGTTTTTGAAGATGCTGATATTGATAAAGCTGTTGAATGGGCTATGTTTGGAGCGTTCTGGACAAATGGCCAGATCTGCAGCGCCACATCACGACTATTATTGCAGGAGAGTATTGCTGATGAATTCTTAAAGAAGATAGCGTCATGGGCTTCATCCATCAAGGTCTCGGATCCACTGGAGAAAGACTGCCGATTGGGACCGCTTGTTAGTGAGAACCAGTACAAGAAGGTGAAAGAGTTCGTGAGAGTGGCGCAAGAAGAGGGAGCCACCCTTGTCTGCGGCGGCAAGAGACCAGATCATTTGTCAAAGGGGTACTTCCTTGCACCGACTGTTCTTTCCAACGTAAAACGCGACATGCAAATCTGGGCGGAGGAGGTGTTTGGTCCAGTCTTAGCTGTCTCCACTTTCAAAACGGAAGAGGAAGCGGTTCTAATGGCAAATGACACTCAGTATGGGCTTGGTGGTGCTGTTATCTCGAAGGATGACGAAAGGTGTAAGCGCGTCTCGGAGGCCTTAGAATGTGGCATTGTATGGATCAACTGTTCCCAACCCTGTTTCTGCCAAGCTCCCTGGGGTGGTAATAAGCGCAGTGGCTTTGGACGAGAGTTGGGAGAATGGGGTCTAGAGAACTATCTCACGGTGAAGCAGATCACCCGCTACATCTCAAACGATGATTGGGGCTGGTATCCCAAGCCCTCAAAACTGTAA

SEQ ID NO.3 ScALDH11A1 nucleotide sequence

ATGGCGGGCCAGGGGTTTTTCCAGGACATTCTGGACGGCGATGTGTTCAAGTTCTACGCCGATGGCGAGTGGAAGACGTCCACCTCCGGAAAGTCTGTCGGCATCACCAACCCTTCTACCTTGAAGACGGCATTCAAAGTGCAAGCGTGCACTCAGGATGAGGTGAACAAGGCCATTGAGAGTGCCAAGGTGGCGCAGAAGGCGTGGGCCAAGACGCCGCTGTGGAAGAGGGCGGAGGCGCTGCACCGGTTCGCAGGCATCCTCAAGGACCAGAAGAACGTCATCGCCGAGTGCCTAGTGAAGGAGGTCGCCAAGGCCCAGAAAGACTCGGTCACTGAGGTGGTGAGGTCTGGTGATTTGATCTCGTACTCAGCCGAGGAGGGCATCAGAATCATGGCCGAGGGCAAGTTCTTGGCCTCGGACAGCTTCCCAGGAAACGGCAGAAACAAGTACTGCCTTGCATCAAAGACTCCACTTGGGGTGGTTCTATGCATCCCTCCCTTCAACTACCCTGTGAACCTGGCTGTGTCCAAGATCGCTCCAGCTCTCATCACAGGAAACGCCGTCATTCTGAAGCCTCCCACTCAGGGTGCCGTGTCGGCCTTGCACATGGTACATTGCGCTCACATGGCTGGCTTTCCCAAGGGCTTGATTGCTGCCGTCACAGGGAAGGGCTCTGAAATCGGAGACCTGCTCACCATGCACCCGGGAATCGACTGCATCAGTTTCACAGGTGGTGACACGGGTATCGCGATTTCGAAGAAGGCAGGCATGATCCCACTTCAGATGGAGCTGGGAGGCAAAGATTGCTGCATTGTTCTGGAGGATGCGGACCTTGAACTTGCCGCCAACAACGTCATCAAGGGCGGATACTCCTACAGTGGGCAACGGTGCACGGCAATCAAGGTGATCTGTGTGATGGAGTCGGTGGCGGAGGAGCTGGTGAAGAACATTGTGGACAAGATGGCCAAGCTCAAGGTGGGAATGCCGGAGGACAACTGTGACATCACTCCCGTTGTCAGCCAGTCCTCCGCGAATTACATCCAGGGACTTGTGGAAGATGCCCAGGCCAAGGGCGCCAAGTTCCATCAGGAATGGAAGAGGGAGAACAACTTGATCCATCCACTTCTGATTGACAATGTGACCCCGGACATGCGAATTGCGTGGGAGGAGCCCTTTGGCCCTGTCATCCCCGTCATCCGCATCAAGACTGTGGAGGAGGGCATCCATCACTGCAATGCCAACAACTTCGCACTTCAGGGGTGCGTCTTCACAAAGGATATCAACAAGGCGATTTTGGTCAGTAACGCTATGGAGTCTGGAACTATTCAAATCAACGCCGCTCCTGCCCGAGGGCCTGACCATTTCCCGTTCCAGGGTCTGCGAGACAGTGGAATTGGCTCACAAGGAGTGACCAACAGTATTCTGATGATGACGAAGGTGAAGTCTACAGTGATCAACCTGCCTGTGGAATCATACACCATGGGTTAA

The size of the amino acid sequence corresponding to the nucleotide sequence is ScALDH2B2 is 527aa, ScALDH10A1 is 506aa, ScALDH11A1 is 496aa, and the amino acid sequence of the corresponding protein is shown in SEQ ID NO. 4-6;

amino acid sequence of ScALDH2B2 protein SEQ ID NO.4

MAMRAMSRKLLSRALRPAAVSRSYGAAAAAAEELGSPMKAPVDVKLTQLLIDGEFVDAASGKTFGVIDPRSEQVIAEVAEGDAEDVNRAVRAARKAFDHGPWPKMPAHKRGEILLRYADLLDQHADELAALETMDSGKPYEQARYGEVPMVSRQFRYYAGWADKIYGTTGPSDGSHAVHTLHEPLGVVGQIIPWNFPIVMYCWKVAPALATGNCVVLKTAEQTPLSALLAGKLALEAGTPPGVLNIVSGYGPTAGAAIAEHMDVDKVAFTGSTEVGKLVMGAAARSNLKPVTLELGGKSPMIVCEDADVDAAVELAHFAIFFNQGQCCCAGSRTFVHESIYDEFVEKSKARALKRVVGDPFRSGVEQGPQVNKEQFNKVLSYIESGQQQGANLLTGGGRLGNVGYYIKPTIFTDVKDGMKIFDEEIFGPVQTIAKFKTLEDVVQRANNTVYGLAAGIFSNNLNTVNTLSRALRAGTVWVNCFDVFDATIPFGGYKQSGIGREKGKEALDNYTQVKAVVTPIHNPAWL

Amino acid sequence of ScALDH10A1 protein SEQ ID NO.5

MGLHAEIVPQRRLFIDGDWVQPRQGKRIPIVNPTTEESVGDIPAATSEDVYAAVKAAKEALHRNNGKDWSKATGKHRATYLRAIAKRVAERKDELAKLESIDCGKPLDEAAWDMDDVSGCFEYYADLAEKLDERQYAPLELPMEQFKCNILRQPVGVVALITPWNYPLLMATWKVAPALAAGCTAILKPSEIASVTCLELASIAKEVGLPNGVLNVITGYGQEAGAPLASHQDVNKVAFTGSTDTGRSIMSAASQLIKPVTLELGGKSPIIVFEDADIDKAVEWAMFGAFWTNGQICSATSRLLLQESIADEFLKKIASWASSIKVSDPLEKDCRLGPLVSENQYKKVKEFVRVAQEEGATLVCGGKRPDHLSKGYFLAPTVLSNVKRDMQIWAEEVFGPVLAVSTFKTEEEAVLMANDTQYGLGGAVISKDDERCKRVSEALECGIVWINCSQPCFCQAPWGGNKRSGFGRELGEWGLENYLTVKQITRYISNDDWGWYPKPSKL

Amino acid sequence of ScALDH11A1 protein of SEQ ID NO.6

MAGQGFFQDILDGDVFKFYADGEWKTSTSGKSVGITNPSTLKTAFKVQACTQDEVNKAIESAKVAQKAWAKTPLWKRAEALHRFAGILKDQKNVIAECLVKEVAKAQKDSVTEVVRSGDLISYSAEEGIRIMAEGKFLASDSFPGNGRNKYCLASKTPLGVVLCIPPFNYPVNLAVSKIAPALITGNAVILKPPTQGAVSALHMVHCAHMAGFPKGLIAAVTGKGSEIGDLLTMHPGIDCISFTGGDTGIAISKKAGMIPLQMELGGKDCCIVLEDADLELAANNVIKGGYSYSGQRCTAIKVICVMESVAEELVKNIVDKMAKLKVGMPEDNCDITPVVSQSSANYIQGLVEDAQAKGAKFHQEWKRENNLIHPLLIDNVTPDMRIAWEEPFGPVIPVIRIKTVEEGIHHCNANNFALQGCVFTKDINKAILVSNAMESGTIQINAAPARGPDHFPFQGLRDSGIGSQGVTNSILMMTKVKSTVINLPVESYTMG

The invention relates to an extreme dry-tolerant erythroaldehyde dehydrogenase gene and a coding protein thereof, wherein the gene is ScALDH2B2, ScALDH10A1 or ScALDH11A1, and the specific method comprises the following steps:

taking a wild collected robust plant of Erythrophlia dentata, soaking the plant in distilled water (but not soaking the whole plant) in a laboratory, recovering for 24 hours, then washing the plant clean, cutting off rhizoid, removing excess water on the surface of the plant, and storing a sample in a refrigerator at the temperature of-80 ℃ for RNA extraction. Cloning coding regions of ScALDH2 and ScALDH10 genes and cloning ScALDH11 genes in a homologous way by an RACE technology according to a odontoid erythroptera transcriptome database; respectively named as ScALDH2B2, ScALDH10A1 and ScALDH11A 1; the lengths of the gene coding regions are 1584bp, 1521bp and 1491bp respectively;

treating a field collected phythrix denticulata plant subjected to rehydration for 24 hours with 100 mu M abscisic acid (ABA), and detecting that 3 ALDH genes all respond to the abscisic acid by using a fluorescent quantitative RT-PCR technology and belong to ABA dependent type;

taking a field collected Erythrina denticulata plant which is rehydrated for 24 hours, drying the plant with silica gel, treating the plant with 250mM NaCl at a low temperature of 4 ℃, and detecting by using a fluorescent quantitative RT-PCR technology to obtain 3 ALDH genes which respond to three abiotic stresses of drought, high salt and low temperature, wherein the response modes are different.

The genes of the extreme dry-tolerant erythroaldehyde dehydrogenase gene and the coding protein thereof, namely ScALDH2B2, ScALDH10A1 and ScALDH11A1 are respectively constructed to a prokaryotic expression vector pET26 to transform prokaryotic cells DE3, and a prokaryotic expression system is used for proving that the 3 aldehyde dehydrogenase genes have the functions of drought resistance and salt tolerance.

The three proteins ScALDH2B2, ScALDH10A1 and ScALDH11A1 are 527aa, prokaryotic expression and purification are carried out, and the recombinant protein substrate enzyme activity experiments prove that ScALDH2B2, ScALDH10A1 and ScALDH11A1 are used as enzymes of a fatty aldehyde dehydrogenase/reductase family, and ScALDH10A1 can also be used as a dehydrogenase/reductase of aromatic aldehyde.

Compared with acceptor plants, the Arabidopsis functions of the transgenic ScALDH2B2, ScALDH10A1 and ScALDH11A1 genes are improved in drought resistance and salt resistance.

The invention clones three genes of extreme xerostomia-resistant erythroaldehyde dehydrogenase family genes ScALDH2B2, ScALDH10A1 and ScALDH11A1 for the first time, the expressions of the three genes can be induced by ABA, drought, high salt and low temperature, but the enzymatic substrates of the three genes are different. When they are expressed in great amount in prokaryotic system and Arabidopsis thaliana, the drought-resisting and salt-tolerant functions of the transformant can be specifically enhanced. Therefore, the three genes have great potential to be applied to the cultivation of transgenic crops with abiotic stress resistance, thereby improving the yield of the crops.

The embodiment of the invention explains the cloning, functional verification, application and research processes of three genes of ScALDH2B2, ScALDH10A1 and ScALDH11A1 and the characteristics of the genes.

Drawings

FIG. 1 shows the total RNA of erythropolis (a), the gene RACE-PCR (b) of erythropolis and the full-length ORF clone (c), Lane M, DL2000 marker; lane1,2,6 sclaldh 2;

lane

3,4,7ScALDH 10; lane 8, ScALDH 11.

FIG. 2 is a comparison of the erythropolis dehydrogenase of the present invention and the ALDH protein from several different species, wherein: at: arabidopsis thaliana; and Zm: corn; hv: barley; and Os: rice; pp: physcomitrella patens; cr: chlamydomonas reinhardtii; EH: salt mustard; sp: spinach; sl: tomatoes; md: apple, apple; and (C) Sc: erythrosine.

FIG. 3 is a response diagram of transcriptional levels of the ScALDH2B2, ScALDH10A1 and ScALDH11A1 genes of 3 erythropolis in accordance with the present invention under conditions of ABA abscisic acid, silica gel drying, 250mM NaCl, low temperature treatment at 4 ℃ for 0h,0.5h,2h,6h,12h and 24 h.

FIG. 4 is a graph of the effect of mannitol and high salt of the invention on the growth of pET-ScALDH/DE3, wherein (a)500mM mannitol was stressed in culture; (b)500mM NaCl was stress cultured.

FIG. 5 shows the specific enzyme activity assay of ScALDH (substrates: hexanal, octanal, trans-hexenal, hexanal and nonanal).

FIG. 6 shows the construction of the transgenic ScALDH Arabidopsis vector and the identification of the transgenic strain, wherein (a) PCR identification of pCAMBIA1301-ScALDH/DH5 alpha bacterial liquid, (b) double digestion identification of pCAMBIA1301-ScALDH plasmid, (c) hygromycin screening of T0 transgenic Arabidopsis (arrows and circles indicate true leaves and roots of the transformed strain, respectively), (d) genome PCR of the transgenic strain and (e) RT-PCR identification; lane M, DL2000 marker;

lanes

1 and 4 are pCAMBIA1301-ScALDH2B 2;

lanes

2 and 5 are pCAMBIA1301-ScALDH10A 1; lane 3,6pCAMBIA1301-ScALDH11A 1; lane 7 is pCAMBIA 1301; lanes 8-10 are the ScALDH2B2 transformants; lanes 11-13 represent the ScALDH10A1 transformant; lanes 14-16 are the ScALDH11A1 transformants; lane 17 is wild type arabidopsis control.

FIG. 7 is a phenotype diagram of 1/2MS medium containing 0(a), 150mM NaCl (B) and 200mM mannitol (c) for 2 strains of ScALDH transgenic Arabidopsis thaliana of the present invention, WT + A2, ScALDH2B2 strain; WT + A10, ScALDH10A 1; WT + A11, ScALDH1A1 strain; WT, wild type.

FIG. 8 is a phenotype map of transgenic Arabidopsis thaliana stressed by 400mM mannitol under the condition of soil culture according to the present invention, A2, and ScALDH2B 2; a10, ScALDH10A 1; a11, ScALDH1A1 strain; WT, wild type control.

Detailed Description

The invention is further illustrated in the following examples, without being restricted thereto.

In the following examples, the experimental procedures not specifying the specific conditions were generally carried out according to the conventional experimental procedures, such as those described in molecular cloning, A laboratory Manual (third edition) (Sambrook J, Russell DW, Janssen K, Argentine J. Huangpeyer et al, 2002, Beijing: scientific Press), or according to the conditions recommended by the manufacturers.

Example 1

Cloning and sequence analysis of 3 genes of ScALDH2B2, ScALDH10A1 and ScALDH11A1 in erythropolis denticola:

1.1 extracting erythrosine RNA and designing a primer: comparing and searching targeted gene sequences (ScALDH2B2, ScALDH10A1 and ScALDH11A1) in NCBI by using aldehyde dehydrogenase EST sequence information in a odontolerythematodes transcriptome database, and conjecturing the deletion direction of ORF and the size of a deleted fragment by combining NCBI and ALDH gene ORF provided in the literature (the deleted fragment information is shown in 1);

ScALDH family gene primer design: based on the sequence information of the aldehyde dehydrogenase gene obtained from the erythroptera dentata transcriptome, the Primer design software Primer primier5.0 was used to design the ORF primers for the sclaldh 11a1 gene and the 5' primers (two each) for the sclaldh 2B2 and the sclaldh 10a1, respectively, to obtain the full sequences, and the Primer sequences were as follows (table 1):

TABLE 1 list of primers used for cloning of the Burseradil dehydrogenase Gene

Note: n indicates that the complete ORF is present in the gene without RACE cloning.

Extracting erythrosine RNA: the collected erythrodons denticulata material is provided with sufficient moisture to keep a sample in a fresh state, sand and soil are cleaned, the sample is washed by distilled water, the surface moisture is sucked, and the sample is quickly frozen by liquid nitrogen and stored at the temperature of minus 80 ℃ for later use; sterilizing glassware such as mortar, beaker, graduated cylinder, and triangular flask for RNA extraction at 180 deg.C for 6h, taking out 0.05g sample from the gun head, EP tube and PCR tube without RNase, grinding with liquid nitrogen, adding appropriate amount of extractive solution, and performing the steps according to the instruction (OMEGA, product number R6827);

preliminarily judging the integrity of the extracted RNA through gel electrophoresis, simultaneously detecting the purity and the content of the RNA by using a nucleic acid quantifier NanoDrop2000(Thermo), and when OD260/OD280 is 2.0 +/-1 and OD260/OD230 is more than 2.0, using an RNA sample for subsequent reverse transcription;

synthesis of erythrodentis cDNA (for full-length ORF cloning): carrying out reverse transcription by using 1 mu g of total RNA at the temperature of 37 ℃ for 45 min; the temperature is 85 ℃ and 5 s; temperature 4 ℃ and ∞ and reaction system as follows (PrimerScript RT-PCR, TAKARA):

synthesis of 5 'and 3' RACE cDNA for Erythrophytic (for cloning ORF deletion genes): the synthesis and procedure of the 5 'and 3' RACE cDNA first strand was performed with reference to the product description (Clontech, cat # PT 4096); storing the cDNA product at-20 deg.c;

1.2 cloning and sequence analysis of the target fragment:

the general RT-PCR and RACE PCR Amplification methods were performed with reference to TAKARA's PrimerScript RT-PCR and SMARTer RACE cDNA Amplification kit, respectively; detecting the PCR amplified fragment by agarose gel electrophoresis, and recovering a PCR product with an expected size by an agarose gel recovery kit; after purifying the product, attaching to a PMD19-T carrier (operating according to the method of the specification); transforming the competence of escherichia coli DH5 alpha, and screening positive clones by a blue-white spot and an Amp antibiotic LB solid plate; activating positive monoclonal through a liquid LB + Amp culture medium, extracting plasmids, and carrying out plasmid PCR identification, wherein a PCR system is as follows:

the PCR reaction condition is pre-denaturation at 94 ℃ for 4 min; the temperature is 94 ℃ for 30s, the temperature is 58 ℃ for 30s, and the temperature is 72 ℃ for 90s, and 30 cycles are operated; finally, extending for 10min at 72 ℃; and (3) sending the plasmid which is identified as positive by PCR to Beijing Huada company for sequencing, simultaneously storing corresponding bacterial liquid into LB + Amp liquid culture medium containing 50% sterile glycerol, quickly freezing by liquid nitrogen, and storing in a refrigerator at-80 ℃. Based on the obtained complete ORF sequence information, primers are designed, the full length of the gene is amplified by RT-PCR technology (shown in figure 1), and the analysis of the sequencing result shows that: the ORF of ScALDH2B2(Unigene 80258) is 1584bp, the ORF of ScALDH10A1 is 1521bp, the ORF of ScALDH11A1 is 1491bp, and the nucleotide sequence is shown in SEQ ID NO 1-3;

SEQ ID NO.1 ScALDH2B2 nucleotide sequence

SEQ ID NO.2 ScALDH10A1 nucleotide sequence

SEQ ID NO.3 ScALDH11A1 nucleotide sequence

The amino acid sequence is shown as SEQ ID NO: 4-6;

amino acid sequence of ScALDH2B2 protein SEQ ID NO.4

Amino acid sequence of ScALDH10A1 protein SEQ ID NO.5

Amino acid sequence of ScALDH11A1 protein of SEQ ID NO.6

And (5) performing biological information analysis on the sequencing sequence. The software used was: performing sequence proofreading and splicing on DNAMAN and PrimierPrimer 5.0 to obtain a full-length cDNA sequence; NCBI/Blast, NCBI/ORF finder evaluation gene ORF; EBI multiple sequence on-line alignment programs ClustalW2(http:// www.ebi.ac.uk/Tools/msa/ClustalW2/) and MEGA6 were used for multiple sequence alignment analysis. The databases used were: protein family proofreading was performed on the GenBank sequence database (GenBank + EMBL + DDBJ + PDBsequesces). The results show (as shown in fig. 2): most erythropolis scoldh are closest in relatedness to physcoldh, a plant of the same moss, and the relatedness between mosses is relatively close to that of other plants, consistent with the fact that they divergently originate from a common ancestor.

Example 2

Analysis of expression patterns of 3 ALDH family genes:

completely rehydrating erythrosine cortex at room temperature (25 + -2 deg.C) for 24 hr, separating erythrosine gametophyte, cleaning, and adding ddH₂Flushing for 3-4 times by using O; absorbing surface water, sampling after abiotic stress and ABA stress; during sampling, the rhizomes were removed and the green shoot bodies (about 1cm) were retained for RNA extraction, and the samples were stored at a temperature of-8 deg.CStoring in a 0 deg.C refrigerator for later use;

drought treatment: uniformly placing 0.5g of completely hydrated moss in a culture dish, placing in a silica gel closed container, and sampling for 10min,0.5h,2h,6h,12h and 24h respectively;

salt treatment: 0.5g of fully hydrated moss is uniformly placed on 250mM NaCl pre-treated filter paper and put into a dish containing 250mM NaCl solution (the gametophyte of the moss cannot be immersed, and the filter paper is kept immersed in the solution during treatment), and samples are taken for 0h,0.5h,2h,6h,12h and 24h respectively;

and (3) cold treatment: uniformly placing 0.5g of completely hydrated moss in a plate, placing in a chromatography cabinet at 4 ℃, and sampling for 0h, 10min,0.5h,2h,6h and 12h respectively;

ABA treatment: 0.5g of fully hydrated moss is uniformly placed on 100 mu M ABA pretreated filter paper and placed in a dish containing 100 mu MABA solution (the gametophyte of the moss cannot be immersed, and the filter paper is kept immersed in the solution during treatment), and sampling is carried out for 0h,0.5h,2h,6h,12h and 24h respectively;

total RNA extraction:

the total RNA extraction using RNAioso reagent (TAKARA company, Dalian connection), the specific steps according to the instruction; the integrity of total RNA was analyzed by agarose gel electrophoresis, and the purity and concentration of total RNA were analyzed by a nucleic acid analyzer (Nanodrop 2000, Thermo Co.). The total RNA sample with better integrity and purity can be used for downstream reverse transcription experiments;

plant RNA is reversely transcribed into cDNA and is subjected to reverse transcription by a reverse transcription kit of TaKaRa company; synthesis of Burnery erythrosine cDNA: total RNA was reverse transcribed at 1. mu.g in the following reaction scheme (PrimerScript RT-PCR, TAKARA):

reverse transcription conditions: the temperature is 37 ℃ and 30 min; the temperature is 85 ℃ and 5 s; temperature 4 ℃ and infinity;

designing a primer:

primer design is carried out by adopting Primer 5.0 software, the length of an amplified fragment is between 100 and 300bp, the annealing temperature is 60 +/-2 ℃, and the general principle of RT-qPCR Primer design is followed. Alpha-tubulin gene is screened out in the laboratory as the most suitable reference gene of the erythrosine, the specificity of the primer is judged through qPCR dissolution curve and agarose gel electrophoresis, the amplification efficiency of the primer is judged through a standard curve, and the annealing temperature of the primer is determined, wherein the used qPCR primer sequences are as follows (Table 2);

TABLE 2 quantitative primer sequences for the genes of the erythroaldehyde dehydrogenase family

Real-time quantitative RT-PCR analysis:

for real-time quantitative RT-PCR, SYBR Premix ExTaq from TaKaRa was used^TMII (perfect read time) kit. The experimental steps are carried out according to the kit instruction, the qPCR system is 20 mu L, and the repetition is carried out for 3 times; PCR amplification was performed with a CFX96 real-time fluorescence quantifier (Bio-Rad, USA); the PCR procedure was: the temperature is 95 ℃ and 30 s; the temperature is 95 ℃ and 5 s; the temperature is 60 ℃, and 30s are circulated for 40 times; the dissolution curve was then added for analysis: the temperature is 65-95 ℃ (30s are gradually increased to 95 ℃, and a fluorescence signal is collected every 0.5 ℃; processing the fluorescence quantitative data by using a fluorescence quantitative instrument with analysis software, calculating according to a NormalizedExpression (delta. CT) method, taking a completely rehydrated sample as a reference, calculating the relative expression quantity of the ScALDHs gene at different time points after the treatment by using an internal reference gene alpha-tubulin expression calibration, and drawing by using Sigmaplot 10.0 software;

under cold stress (4 ℃), the expression of the ScALDH10A1 is up-regulated, and the expression amount of the ScALDH10A1 has accumulation peak at 0.5h and 24h under cold stress, and is about 10 times of that of a control; ScALDH2B2 quickly up-regulated in response to cold stress for about 0.5h, the expression level is about 5 times that of a control, and then the expression level is reduced; sclaldh 11a1 responded poorly in cold stress (fig. 3); under salt stress, the expression level of ScALDH11A1 is about 5 times that of a control (figure 3); under drought stress, the expression level of the ScALDH2B2 is more than 6 times that of the control; the response of ScALDH2B2 is 12h after the stress, so that the response is delayed; the other genes all have weak up-regulation expression (FIG. 3); ABA is important for plants to resist abiotic stress, under the treatment of hormone ABA, ScALDH11A1 is up-regulated to respond to ABA induction, the expression level is more than 4 times of that of a control, and the results show that ALDH gene regulation pathways are different (figure 3).

Example 3

Prokaryotic expression of ScALDH and influence thereof on drought resistance and salt tolerance of escherichia coli:

vector construction and transformation:

designing primers with enzyme cutting sites according to the ORF full length (ScALDH2B2, ScALDH7B1, ScALDH10A1, ScALDH11A1 and ScALDH21A1) of the aldehyde dehydrogenase gene obtained in the early stage, wherein the upstream primer considers a correct reading frame, and the downstream primer removes a stop codon to fuse the upstream primer with a His tag; the primer sequences were as follows (table 3):

TABLE 3 prokaryotic expression primers for the Burseradilla dehydrogenase gene

Note: the restriction enzyme site sequence is underlined.

Carrying out PCR amplification by taking a clone bacterial strain with correct sequencing of the target gene stored in the previous period as a template, connecting an amplification product to a pMD18-T vector, and randomly selecting positive clone sequencing; recovering the enzyme digestion product through a correct target gene fragment and a PET26b (+) plasmid by double enzyme digestion sequencing; connecting the target fragment and the vector through T4DNA ligase to obtain a fusion prokaryotic expression vector; after transformation to DH5 alpha competence, selecting positive clone for sequencing, then transforming the plasmid with correct sequencing to escherichia coli BL21(DE3) competence by adopting a heat shock method, and obtaining a prokaryotic fusion expression strain with aldehyde dehydrogenase gene;

effect of the sclaldh gene on the stress resistance of escherichia coli:

selecting a prokaryotic fusion expression strain with an aldehyde dehydrogenase gene, adding the prokaryotic fusion expression strain into 5mL LB liquid medium containing kanamycin, carrying out overnight shaking culture at 37 ℃, extracting plasmids, and detecting positive clones by bacterial liquid PCR; diluting the inoculated strain into a fresh LB culture medium according to the proportion of 1:100, carrying out shake culture at the temperature of 37 ℃ until the OD600 is about 0.6, adding 1mM IPTG to induce the target protein to express, and continuing the shake culture for 2.5 h; taking 1mL of bacterial liquid, centrifuging at 12000rpm for 5min, discarding the supernatant, then resuspending the thalli by using 30 mu L of freshly prepared electrophoresis loading buffer solution, boiling for 10min, centrifuging at 12000rpm for 10min, taking 20 mu L of supernatant, carrying out SDS-PAGE electrophoresis, and verifying protein bands successfully expressed by 3 ScALDH genes in prokaryotic cells;

taking out the aldehyde dehydrogenase prokaryotic expression strain frozen at the temperature of minus 80 ℃ from a refrigerator, carrying out line drawing inoculation on a solid LB + Kan plate, and culturing overnight at the temperature of 37 ℃; selecting a single colony, inoculating the single colony into an LB + Kan liquid culture medium, performing shake overnight culture, inoculating the single colony into a fresh LB + Kan liquid culture medium according to a ratio of 1:100, performing shake activation culture at 37 ℃ for 3h until OD600 is 0.6-0.8, and adding 1mM isopropyl-beta-D-thiogalactoside (IPTG) to continue induction culture for 2.5 h;

stress in solid medium: after gradient dilution, 5 mu L of bacterial liquid is respectively spotted in non-stress culture media and stress culture media (NaCl salt stress and mannitol drought stress), cultured at the temperature of 37 ℃, the size of bacterial plaque is observed, and the experiment is repeated for 3 times;

the influence of the ScALDH gene on the stress resistance of the Escherichia coli is judged by detecting the colony number and the plaque size of the Escherichia coli by adopting the methods of osmotic stress and salt stress, as shown in figure 4, on LB culture medium containing 500mM mannitol and NaCl, 10 dilutions are respectively shown^-3And 10^-4Under the double condition, only sporadic bacterial plaques are seen in a control pET26/DE3, but obvious bacterial colonies can still be seen in escherichia coli expressing the aldehyde dehydrogenase gene; this shows that prokaryotic expression of aldehyde dehydrogenase can improve the osmotic stress resistance and salt tolerance of Escherichia coli. However, the improvement effect of different aldehyde dehydrogenases is markedly different.

Example 4

Purification and enzyme activity analysis of the ScaLDH family protein:

preparation of crude enzyme solution of ScALDH protein:

in order to obtain the protein activity and ensure the protein yield, the optimized expression conditions of the ScaLDH-His protein are as follows: culturing the strain overnight at 18 deg.C (1:100 inoculation), inoculating the strain at 1:50, activating the strain to OD600 of 0.6, and inducing at 18 deg.C for 15h (OD600 of about 1.3) with IPTG concentration of 0.25 mM; centrifuging at 4 deg.C and 5000rpm for 15min to collect thallus; resuspend the cells with phosphate buffered saline PBS (250mL of cells were resuspended with 10mL PBS); placing the bacteria on ice, breaking cell wall with ultrasonic wave (ultrasonic for 3s, interval for 3s) for 30min, and maintaining low temperature (ice-water mixing temperature to prevent heating of bacteria liquid); centrifuging at 4 ℃ and 13000rpm, collecting supernatant as initial enzyme solution, and judging the expression quantity of the target protein in the crude enzyme solution by SDS-PAGE electrophoresis;

and (3) purifying the crude enzyme solution of the ScALDH protein:

ni Sepharose of GE^TMStoring the 6Fast Flow protein purification medium in 20% ethanol, mixing uniformly, directly taking 1.0mL to 2mL centrifuge tubes, and centrifuging for 5min at 500 Xg; discarding the supernatant, adding 2mL of deionized water, gently shaking, uniformly mixing, and centrifuging; the supernatant was discarded, 2mL of binding buffer was added, mixed well and centrifuged. Discarding the supernatant, adding 1mL of binding buffer solution, uniformly mixing, and transferring to a protein purification column;

balancing the column with 5-10 times column volume of PBS solution, adding crude protein sample into treated nickel column, purifying protein in chromatography cabinet at 4 deg.C, and mixing filler and protein in multifunctional rotary table (360 deg.) for 4 hr or overnight; after the filler and the protein are fully combined and naturally settled, eluting the hybrid protein by PBS (imidazole concentration gradient: 20Mm, 60mM, 80mM, 100mM, 150mM and 200mM) with different concentration gradients of imidazole in 10 times of column volume, and respectively collecting effluent for subsequent SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) detection and determining the elution concentration of the target protein; finally, the residual protein in the packing was washed with 250mM PBS, and the regeneration of the packed column was performed as described (5 volumes of deionized water, 5 volumes of elution buffer, and 10 volumes of binding buffer);

detecting the effluent containing the target protein by SDS-PAGE, and performing subsequent protein concentration and buffer replacement steps, wherein the method I comprises the following steps: loading 10mL of purified solution containing target protein into treated dialysis bag, fixing and suspending in beaker containing 500mL of Tris. Cl (0.1M, pH8.0), magnetically stirring, dialyzing in chromatography cabinet at 4 deg.C, changing dialysis buffer every 4-6h, and repeating for 3 times; concentrating the protein purification solution after liquid replacement by using an ultrafiltration tube according to the instructions of ultrafiltration tube products; the obtained pure protein is stored in crushed ice, and simultaneously, a target protein band is detected by SDS-PAGE, and the target protein concentration is detected by a Bradford method; the second method comprises the following steps: directly diluting 10mL of purified solution containing target protein to 50mL by using enzyme reaction buffer Tris.Cl (0.1M, pH8.0), and then performing solution replacement operation according to the specification of an ultrafiltration tube product;

3 ScaLDH protease enzymatic Properties:

determination of aldehyde dehydrogenase Activity:

protein content determination: measuring protein content by Coomassie brilliant blue method, preparing standard curve by product specification method with Bovine Serum Albumin (BSA) as standard, reacting for 5min, measuring absorbance wavelength at 595nm, and drawing standard curve.

Drawing an NADH standard curve: the NADH solution concentration is 3mM, 0, 1, 3, 5, 7, 9 and 11 mu L NADH (nicotinamide adenine dinucleotide) solution is respectively sucked, diluted to 200 mu L by Tris.Cl, and the absorbance value at OD340nm is measured, thereby drawing a standard curve;

the ALDH activity was defined with reference to the Sigma-aldrich website (http:// www.sigmaaldrich.com/content/dam/Sigma-aldrich/docs/Sigma/Bulletin/1/mak082bul. pdf) and the aldehyde dehydrogenase (ALDH) activity was defined as the temperature 30 ℃ and pH8.0, and the amount of enzyme used to produce 1. mu. mol NADH per minute was one enzyme activity unit (U); the ALDH Activity formula is B multiplied by Sample Diluenfactor/(Reaction Time)/V; b is NADH production (nmole) in Reaction Time, Reaction Time is Tfinal-tinitial (minutes); v is the sample volume (mL), ALDH activity reported as nmole/min/mL is milliunit/mL, and specific enzyme activity is enzyme activity/protein amount.

Enzyme reaction system (200 μ L): aldehyde substrate 0.2-5uL, 1mmol/L NAD⁺0.1mol/L Tris.Cl (pH8.0), 0.1. mu.g of purified enzyme solution, and measuring the change in absorbance at 25 ℃ and 340 nm;

whether the enzyme reaction proceeded was checked by HPLC and mass spectrometry: sampling the reaction product, and respectively carrying out HPLC (high performance liquid chromatography) and mass spectrometry on the components of the reaction mixed solution;

substrate specificity of the enzyme:

selecting fatty aldehyde (formaldehyde, n-octanal, trans-2-hexene-1-aldehyde, hexanal, nonanal, acetaldehyde) and aromatic aldehyde (salicylaldehyde) and aromatic aldehyde (o-hydroxybenzaldehyde)Cinnamic aldehyde (phenylacrolein) as substrate with NAD⁺Determining enzyme activity as coenzyme;

relative enzyme activity assay: the aldehyde substrate is taken at 1 μ L, protease 0.1 μ g, temperature 30 deg.C, pH8.0, NAD⁺Reaction at 1mM for 1 min; the enzyme activity taking trans-2-hexene-1-aldehyde as a substrate is calculated by 100 percent;

as can be seen from FIG. 5, the aldehyde substrate was used in an amount of 1. mu.L (formaldehyde, trans-2-hexen-1-al, hexanal, octanal and nonanal), protease 0.1. mu.g, pH8.0 at 30 ℃ and NAD⁺The specific enzyme activity values of three proteins are shown under the condition of 1mM reaction for 1 min: the specific enzyme activity values of the ScALDH10 are all the lowest. The differences between the ScALDH2 and the ScALDH11 were not significant.

Example 5

Drought resistance and salt tolerance analysis of 3 ScALDH genes in Arabidopsis thaliana:

construction of plant overexpression vector and transformation of Agrobacterium:

primers with appropriate cleavage sites were designed according to the sequence of the gene of interest and the vector Multiple Cloning Site (MCS), the sequences are shown in the following table (Table 7), the genes of the erythrosine dehydrogenase obtained in the previous stage are used as templates, and the PCR procedure is as follows: pre-denaturation at 95 ℃ for 3 min; the temperature is 95 ℃ for 30s, the temperature is 58 ℃ for 30s, and the temperature is 72 ℃ for 90s, and 30 cycles are carried out; finally, extending for 7min at 72 ℃, cloning the amplified product to a pMD19-T simple vector, then transforming to escherichia coli DH5 alpha, selecting positive clones, sending the positive clones to Huada gene company for sequencing, preserving the correctly sequenced clones (pMD19-T simple/ScALDH/DH5 alpha), simultaneously extracting plasmids, using restriction enzymes described in the following table to enzyme-cut pCAMBIA1301 and pMD19-T simple/ScALDH plasmids, recovering target fragments, and connecting the target gene and the vector at 16 ℃;

TABLE 4 Burtonia erythroaldehyde dehydrogenase family Gene cloning primer sequences

And after the T4DNA ligase is connected with the two, the two are transformed into escherichia coli DH5 alpha, single colony is selected for bacteria shaking through kanamycin resistance screening, bacteria liquid PCR (figure 6a) is carried out, PCR positive cloning is carried out to extract plasmids for double restriction enzyme identification (figure 6b), positive cloning plasmids with correct sequencing are transformed into agrobacterium EHA105, streptomycin and kanamycin double resistance screening is carried out, and bacteria liquid PCR amplification is carried out by using specific primers to obtain fragments with expected sizes. The construction of the pCAMBIA1301-ScALDH plant expression vector is proved to be successful;

preparing agrobacterium EHA105 competence, then transforming the constructed vector carrying the ScALDH target gene to agrobacterium EHA105, verifying that the transformed strain of the resistance plate positive clone has the exogenous ScALDH target gene through PCR, and storing the agrobacterium EHA105 transformed strain for later use;

agrobacterium-mediated transformation of arabidopsis thaliana:

and (3) culturing Arabidopsis thaliana:

sterilizing with 15% sodium hypochlorite for 5min, shaking to suspend the seeds, washing with sterile water for 4-6 times, slightly rotating and centrifuging to precipitate the seeds, removing supernatant, inoculating to MS or 1/2MS solid culture medium, vernalizing at 4 deg.C for 36 hr, and culturing in a climatic chamber at 22 deg.C for 16 hr under continuous illumination;

transplanting the seedlings growing for 6 days on the solid culture medium into artificial soil, wherein the nutrient soil is as follows: vermiculite (3:1), the preservative film naturally grows after keeping the humidity for one week, and watering is carried out every 3 days to keep the soil humidity;

agrobacterium mediated floral dip transformation of Arabidopsis thaliana:

the arabidopsis transgenic method is an agrobacterium tumefaciens-mediated inflorescence dip-dyeing method, the adopted agrobacterium strain is EHA105, and the transformation receptor material is Columbia wild type arabidopsis; transforming arabidopsis thaliana by inflorescence soaking method, collecting plant with more inflorescences after bolting and good growth condition (selecting inflorescence containing bud to be released), cutting flowering flowers, watering the day before transformation, culturing transgenic Agrobacterium (pCAMBIA1301-ScALDH/EHA105 strain) at 28 ℃ overnight until OD600 value is about 1.2, centrifuging at 5000rpm for 8min, resuspending the strain in freshly prepared transformation liquid (transformation buffer solution components: 5% Sugar + 0.02% Silwet L-77(200 uL/L) +100 uL/L Triton X-100+100 uM AS +2 ng/L6-BA + Agrobacterium, OD600 value is 0.8-1.0, soaking arabidopsis thaliana inflorescences in bacterial liquid for 30s during transformation, slightly shaking the cells during the period, taking out, placing the cells in a room for 10min, staining once again, bagging the transformed plant, transversely placing the plant in a tray to be cultured for about 36h at normal temperature, performing vertical normal culture on the next day, performing dip dyeing once again after one week according to the growth state of the seedlings, harvesting seeds (T0) after culturing for 3-4 weeks, and storing under a dry condition for later use;

t0 seeds were harvested, sterilized and plated on 1/2MS medium containing 70mg/L hygromycin. The wild type can only sprout two cotyledons on a hygromycin culture medium, and dies gradually along with the prolonging of the culture time, and can not grow true leaves and root systems; the resistant seedlings can grow true leaves and root systems in about 10 days and show obvious kanamycin resistance (figure 6 c); extracting genome DNA from the obtained resistant seedlings, and detecting by PCR to obtain T1 generation ScALDH transgenic arabidopsis strains; FIG. 6d shows a part of PCR positive transgenic Arabidopsis plants, further detecting the expression of ScALDH by RT-PCR, the results show that ScALDH is successfully expressed in Arabidopsis (FIG. 6 e);

stress resistance analysis of transgenic ScALDHs arabidopsis thaliana:

detecting transgenic plants and analyzing expression quantity:

T₀the generation seeds are paved on a screening culture medium containing 70 mu g/mL hygromycin, the temperature is 4 ℃, the vernalization is carried out for 36h, the seeds are moved to a climatic chamber, the seeds grow for one week (22 ℃) under the condition of 16h continuous illumination, the screened positive seedlings (capable of growing true leaves on a resistant culture medium) are transplanted into nutrient soil to continue growing, after a preservative film is moisturized for one week, the normal culture is carried out, the seeds are harvested (T1 generation), the T1 generation seeds are inoculated into the hygromycin resistant culture medium for screening, the T2 generation seeds with positive PCR are harvested, and the T3 generation plants are subjected to subculture screening for stress resistance analysis;

harvesting seeds of a single plant: extracting the genome DNA of the resistant plant and the genome DNA of the untransformed plant, performing PCR reaction by using gene specific primers, selecting T3 positive arabidopsis thaliana to extract the total RNA of seedlings, and analyzing the expression condition of the foreign gene in the transgenic plant by using an RT-PCR method;

stress treatment of transgenic arabidopsis thaliana:

the transgenic plants are subjected to stress growth in a culture medium: soaking seeds of T2 generation and wild type seeds in 15% sodium hypochlorite for 3min, washing with sterile water for 4-6 times, sowing on 1/2MS culture medium, vernalizing at 4 ℃ for 2d, vertically culturing in a phytotron with 16h/8h light/dark period illumination at 22 ℃, transferring to 1/2MS (as a control) containing 1/2MS +300mM mannitol and 1/2MS +150mM NaCl solid culture medium after 5-7 days of growth, and vertically culturing at 22 ℃ for 16h continuous illumination; the results show that: (i) after 9 days of salt stress, the growth of transgenic seedlings and WT seedlings is inhibited, the root system growth of wild seedlings is completely inhibited, the root system of a transgenic plant can still continue to grow (figure 7b), and the root length of the transgenic plant is 37-150% of the elongation of the WT root system; (i i) under the stress of mannitol, after 9d of growth, the growth of transgenic seedlings and wild seedlings is inhibited, the leaf number (shown as WT + A10 plants) and the fresh weight of wild seedlings are affected (figure 7c), and the fresh weight of transgenic lines is 41-67% of that of WT plants;

the transgenic arabidopsis thaliana grows under stress in nutrient soil: in order to ensure the accuracy of the experiment and eliminate the influence of the plant growth period on the stress sensitivity, the subsequent experiment takes a month-old soil-cultured seedling as a material to analyze the drought resistance of the ScALDH transgenic plant;

soaking seeds of T2 generation and wild type seeds in 15% sodium hypochlorite for 3min, washing with sterile water for 4-6 times, sowing on 1/2MS culture medium, vernalizing at 4 ℃ for 2d, taking out, vertically placing in an artificial climate chamber with the temperature of 22 ℃,16 h/8h light/dark period illumination for culture, growing for 7 days, planting in nutrient soil (the nutrient soil is vermiculite: 3:1) at the temperature of 22-24 ℃, the relative humidity of 60%, 16h illumination/8 h dark illumination for culture, and covering a film for moisturizing in the first week; after three weeks of growth recovery, the leaves of the wild-type plants are treated with 400mM mannitol solution until wilting (4 days) begins, as shown in FIG. 8, under normal conditions, the phenotypes of the transgenic and wild-type plants have no obvious difference, and when the transgenic and wild-type Arabidopsis are irrigated with 400mM mannitol solution for drought treatment, the leaves of the wild-type Arabidopsis become brown or even withered and yellow after 4 days, and the leaves of the transgenic plants can still keep green.

The above results show that: the ScALDHs transgenic plant has enhanced resistance to drought stress and salt stress, and maintains the normal growth and development process of the plant under the salt stress.

Sequence listing

<110> Sinkiang institute for ecology and geography  of Chinese academy of sciences

<120> extremely drought-resistant erythropolis aldehyde dehydrogenase gene and protein encoded by same

<160>6

<170>SIPOSequenceListing 1.0

<210>1

<211>1584

<212>DNA

<213> Erythrophora dentata (Syntricia caninervis)

<220>

<221>CDS

<222>(1)..(1491)

<400>1

atg gcg atg agg gcg atg agc agg aag ctc ctg tcg cgg gcg ctg cga 48

Met Ala Met Arg Ala Met Ser Arg Lys Leu Leu Ser Arg Ala Leu Arg

1 5 10 15

cct gca gcg gtt agt cga agc tac ggc gcg gca gcg gca gcc gcg gag 96

Pro Ala Ala Val Ser Arg Ser Tyr Gly Ala Ala Ala Ala Ala Ala Glu

20 2530

gag ctg ggg agc ccc atg aag gcg cca gtg gac gtg aag ctc acc cag 144

Glu Leu Gly Ser Pro Met Lys Ala Pro Val Asp Val Lys Leu Thr Gln

35 40 45

ctg ctg atc gac ggg gag ttc gtg gac gcc gcg tcg ggg aag acg ttc 192

Leu Leu Ile Asp Gly Glu Phe Val Asp Ala Ala Ser Gly Lys Thr Phe

50 55 60

ggg gtc atc gac ccc aga agc gag caa gtc atc gcg gag gtc gcc gag 240

Gly Val Ile Asp Pro Arg Ser Glu Gln Val Ile Ala Glu Val Ala Glu

65 70 75 80

ggc gac gcg gag gac gtc aac cgc gcg gtg cgc gct gca agg aag gcg 288

Gly Asp Ala Glu Asp Val Asn Arg Ala Val Arg Ala Ala Arg Lys Ala

85 90 95

ttc gac cat ggg ccg tgg ccg aaa atg ccc gcc cac aag cgc ggc gag 336

Phe Asp His Gly Pro Trp Pro Lys Met Pro Ala His Lys Arg Gly Glu

100 105 110

att ctc ctc agg tat gcg gac ttg ttg gac cag cac gcg gat gag ctc 384

Ile Leu Leu Arg Tyr Ala Asp Leu Leu Asp Gln His Ala Asp Glu Leu

115 120 125

gcg gcg ctg gag acg atg gac tct ggg aag ccg tac gag caa gcg cgc 432

Ala Ala Leu Glu Thr Met Asp Ser Gly Lys Pro Tyr Glu Gln Ala Arg

130 135 140

tac ggg gag gtg ccg atg gtg tcg cgg cag ttc cga tac tac gcg ggg 480

Tyr Gly Glu Val Pro Met Val Ser Arg Gln Phe Arg Tyr Tyr Ala Gly

145 150 155 160

tgg gcg gac aag ata tac ggc acg acg ggg ccg tct gat gga agc cac 528

Trp Ala Asp Lys Ile Tyr Gly Thr Thr Gly Pro Ser Asp Gly Ser His

165 170 175

gcg gtg cac acg ctc cac gag ccg ttg ggt gtg gtg ggg cag atc atc 576

Ala Val His Thr Leu His Glu Pro Leu Gly Val Val Gly Gln Ile Ile

180 185 190

ccc tgg aac ttc ccc atc gtc atg tac tgc tgg aag gtg gcg cct gcg 624

Pro Trp Asn Phe Pro Ile Val Met Tyr Cys Trp Lys Val Ala Pro Ala

195 200 205

ctg gca acg ggg aac tgc gtc gtg cta aag acc gcg gag cag acg ccg 672

Leu Ala Thr Gly Asn Cys Val Val Leu Lys Thr Ala Glu Gln Thr Pro

210 215 220

ctg tcc gcc ctt ctg gcc ggg aag ctg gcg ttg gag gcc ggc att ccc 720

Leu Ser Ala Leu Leu Ala Gly Lys Leu Ala Leu Glu Ala Gly Ile Pro

225 230 235 240

ccg ggc gtg ctc aac atc gtc tcc ggg tac ggc ccc acc gcg ggc gcc 768

Pro Gly Val Leu Asn Ile Val Ser Gly Tyr Gly Pro Thr Ala Gly Ala

245 250 255

gcc ata gcc gag cac atg gac gtc gac aag gtc gcc ttc acg ggc tcc 816

Ala Ile Ala Glu His Met Asp Val Asp Lys Val Ala Phe Thr Gly Ser

260 265 270

acc gag gtc ggg aag ctc gtc atg ggg gcg gca gcg cgc agc aac ttg 864

Thr Glu Val Gly Lys Leu Val Met Gly Ala Ala Ala Arg Ser Asn Leu

275 280 285

aag ccg gtg acg ctg gag ctc gga ggg aag tcg ccc atg atc gtg tgc 912

Lys Pro Val Thr Leu Glu Leu Gly Gly Lys Ser Pro Met Ile Val Cys

290 295 300

gag gac gcg gac gtg gac gct gcc gtg gag ctg gcg cac ttc gcc atc 960

Glu Asp Ala Asp Val Asp Ala Ala Val Glu Leu Ala His Phe Ala Ile

305 310 315 320

ttc ttc aac cag ggg cag tgc tgc tgc gcg ggg tcg cgc acg ttt gtg 1008

Phe Phe Asn Gln Gly Gln Cys Cys Cys Ala Gly Ser Arg Thr Phe Val

325 330 335

cac gag agc atc tac gac gag ttc gtg gag aag tcc aag gcg cgc gcc 1056

His Glu Ser Ile Tyr Asp Glu Phe Val Glu Lys Ser Lys Ala Arg Ala

340 345 350

ctg aag cgc gtt gtg ggc gac cct ttc agg agc ggc gtg gag cag ggc 1104

Leu Lys Arg Val Val Gly Asp Pro Phe Arg Ser Gly Val Glu Gln Gly

355 360 365

cct cag gtc aac aag gag cag ttt aac aag gtc ctc tcg tac atc gag 1152

Pro Gln Val Asn Lys Glu Gln Phe Asn Lys Val Leu Ser Tyr Ile Glu

370 375 380

agc ggg cag cag cag ggc gcc aac ctc ctc aca ggt gga ggt cgc ttg 1200

Ser Gly Gln Gln Gln Gly Ala Asn Leu Leu Thr Gly Gly Gly Arg Leu

385 390 395 400

gga aac gta ggc tac tac atc aag ccc aca atc ttc act gac gtg aaa 1248

Gly Asn Val Gly Tyr Tyr Ile Lys Pro Thr Ile Phe Thr Asp Val Lys

405 410 415

gac ggt atg aag atc ttc gac gag gag atc ttc ggt cct gtg cag acg 1296

Asp Gly Met Lys Ile Phe Asp Glu Glu Ile Phe Gly Pro Val Gln Thr

420 425 430

att gcc aag ttc aaa acc ttg gaa gat gtg gtg cag cgg gcc aac aac 1344

Ile Ala Lys Phe Lys Thr Leu Glu Asp Val Val Gln Arg Ala Asn Asn

435 440 445

aca gtg tac gga ttg gca gcg ggc att ttc agc aac aac ttg aac acg 1392

Thr Val Tyr Gly Leu Ala Ala Gly Ile Phe Ser Asn Asn Leu Asn Thr

450 455 460

gtg aac acg ttg agc cgg gca ctg cga gcg ggc acc gtg tgg gtg aac 1440

Val Asn Thr Leu Ser Arg Ala Leu Arg Ala Gly Thr Val Trp Val Asn

465 470 475 480

tgc ttc gac gtg ttc gac gcc acc att ccg ttc ggg ggg tac aag cag 1488

Cys Phe Asp Val Phe Asp Ala Thr Ile Pro Phe Gly Gly Tyr Lys Gln

485 490 495

agc ggcatcgggc gggagaaggg caaggaggcc ctggacaact acacgcaggt 1541

Ser

gaaggcggtg gtgacgccta tccacaaccc cgcgtggctg taa 1584

<210>2

<211>1521

<212>DNA

<213> Erythrophora dentata (Syntricia caninervis)

<400>2

atgggtcttc acgctgagat tgtgccccag cgccgcctct tcatcgacgg cgactgggtg 60

cagccgcgcc agggcaagcg cattcccatt gtcaatccca ccacggagga gagcgtgggg 120

gacattccag ctgcaacctc ggaagacgtc tatgctgcag tgaaggcggc taaggaagcg 180

ttacatcgca acaatggcaa ggactggtct aaggccactg gaaagcaccg cgcaacctac 240

ctccgagcca ttgccaagag ggtggctgag agaaaagacg agcttgcgaa gctggagtcc 300

atcgactgtg gcaagcctct cgatgaagcg gcgtgggata tggatgatgt atcagggtgt 360

tttgagtact acgcggacct ggcagagaag ttggatgaga ggcagtatgc tcctctggaa 420

cttcccatgg agcaattcaa atgcaacata ttgcgacagc ctgttggagt tgttgctctt 480

atcacaccct ggaattaccc acttttgatg gctacttgga aagttgctcc agcgcttgca 540

gctggatgca ctgccattct gaaaccatct gagattgcct ctgtcacttg cttggagttg 600

gcgagtattg ccaaagaagt tggacttccg aacggagtct tgaatgtgat cactggatat 660

ggacaggaag ctggtgcacc cttggcctca catcaggatg taaacaaggt agcttttact 720

ggaagcacag atactggcag gtctatcatg tccgcggcat cacagctcat caagccagta 780

acattggaat tgggtggaaa gagccccatc atagtttttg aagatgctga tattgataaa 840

gctgttgaat gggctatgtt tggagcgttc tggacaaatg gccagatctg cagcgccaca 900

tcacgactat tattgcagga gagtattgct gatgaattct taaagaagat agcgtcatgg 960

gcttcatcca tcaaggtctc ggatccactg gagaaagact gccgattggg accgcttgtt 1020

agtgagaacc agtacaagaa ggtgaaagag ttcgtgagag tggcgcaaga agagggagcc 1080

acccttgtct gcggcggcaa gagaccagat catttgtcaa aggggtactt ccttgcaccg 1140

actgttcttt ccaacgtaaa acgcgacatg caaatctggg cggaggaggt gtttggtcca 1200

gtcttagctg tctccacttt caaaacggaa gaggaagcgg ttctaatggc aaatgacact 1260

cagtatgggc ttggtggtgc tgttatctcg aaggatgacg aaaggtgtaa gcgcgtctcg 1320

gaggccttag aatgtggcat tgtatggatc aactgttccc aaccctgttt ctgccaagct 1380

ccctggggtg gtaataagcg cagtggcttt ggacgagagt tgggagaatg gggtctagag 1440

aactatctca cggtgaagca gatcacccgc tacatctcaa acgatgattg gggctggtat 1500

cccaagccct caaaactgta a 1521

<210>3

<211>1491

<212>DNA

<213> Erythrophora dentata (Syntricia caninervis)

<400>3

atggcgggcc aggggttttt ccaggacatt ctggacggcg atgtgttcaa gttctacgcc 60

gatggcgagt ggaagacgtc cacctccgga aagtctgtcg gcatcaccaa cccttctacc 120

ttgaagacgg cattcaaagt gcaagcgtgc actcaggatg aggtgaacaa ggccattgag 180

agtgccaagg tggcgcagaa ggcgtgggcc aagacgccgc tgtggaagag ggcggaggcg 240

ctgcaccggt tcgcaggcat cctcaaggac cagaagaacg tcatcgccga gtgcctagtg 300

aaggaggtcg ccaaggccca gaaagactcg gtcactgagg tggtgaggtc tggtgatttg 360

atctcgtact cagccgagga gggcatcaga atcatggccg agggcaagtt cttggcctcg 420

gacagcttcc caggaaacgg cagaaacaag tactgccttg catcaaagac tccacttggg 480

gtggttctat gcatccctcc cttcaactac cctgtgaacc tggctgtgtc caagatcgct 540

ccagctctca tcacaggaaa cgccgtcatt ctgaagcctc ccactcaggg tgccgtgtcg 600

gccttgcaca tggtacattg cgctcacatg gctggctttc ccaagggctt gattgctgcc 660

gtcacaggga agggctctga aatcggagac ctgctcacca tgcacccggg aatcgactgc 720

atcagtttca caggtggtga cacgggtatc gcgatttcga agaaggcagg catgatccca 780

cttcagatgg agctgggagg caaagattgc tgcattgttc tggaggatgc ggaccttgaa 840

cttgccgcca acaacgtcat caagggcgga tactcctaca gtgggcaacg gtgcacggca 900

atcaaggtga tctgtgtgat ggagtcggtg gcggaggagc tggtgaagaa cattgtggac 960

aagatggcca agctcaaggt gggaatgccg gaggacaact gtgacatcac tcccgttgtc 1020

agccagtcct ccgcgaatta catccaggga cttgtggaag atgcccaggc caagggcgcc 1080

aagttccatc aggaatggaa gagggagaac aacttgatcc atccacttct gattgacaat 1140

gtgaccccgg acatgcgaat tgcgtgggag gagccctttg gccctgtcat ccccgtcatc 1200

cgcatcaaga ctgtggagga gggcatccat cactgcaatg ccaacaactt cgcacttcag 1260

gggtgcgtct tcacaaagga tatcaacaag gcgattttgg tcagtaacgc tatggagtct 1320

ggaactattc aaatcaacgc cgctcctgcc cgagggcctg accatttccc gttccagggt 1380

ctgcgagaca gtggaattgg ctcacaagga gtgaccaaca gtattctgat gatgacgaag 1440

gtgaagtcta cagtgatcaa cctgcctgtg gaatcataca ccatgggtta a 1491

<210>4

<211>527

<212>PRT

<213> Erythrophora dentata (Syntricia caninervis)

<400>4

Met Ala Met Arg Ala Met Ser Arg Lys Leu Leu Ser Arg Ala Leu Arg

1 5 10 15

Pro Ala Ala Val Ser Arg Ser Tyr Gly Ala Ala Ala Ala Ala Ala Glu

2025 30

Glu Leu Gly Ser Pro Met Lys Ala Pro Val Asp Val Lys Leu Thr Gln

35 40 45

Leu Leu Ile Asp Gly Glu Phe Val Asp Ala Ala Ser Gly Lys Thr Phe

50 55 60

Gly Val Ile Asp Pro Arg Ser Glu Gln Val Ile Ala Glu Val Ala Glu

65 70 75 80

Gly Asp Ala Glu Asp Val Asn Arg Ala Val Arg Ala Ala Arg Lys Ala

85 90 95

Phe Asp His Gly Pro Trp Pro Lys Met Pro Ala His Lys Arg Gly Glu

100 105 110

Ile Leu Leu Arg Tyr Ala Asp Leu Leu Asp Gln His Ala Asp Glu Leu

115 120 125

Ala Ala Leu Glu Thr Met Asp Ser Gly Lys Pro Tyr Glu Gln Ala Arg

130 135 140

Tyr Gly Glu Val Pro Met Val Ser Arg Gln Phe Arg Tyr Tyr Ala Gly

145 150 155 160

Trp Ala Asp Lys Ile Tyr Gly Thr Thr Gly Pro Ser Asp Gly Ser His

165 170 175

Ala Val His Thr Leu His Glu Pro Leu Gly Val Val Gly Gln Ile Ile

180 185 190

Pro Trp Asn Phe Pro Ile Val Met Tyr Cys Trp Lys Val Ala Pro Ala

195 200 205

Leu Ala Thr Gly Asn Cys Val Val Leu Lys Thr Ala Glu Gln Thr Pro

210 215 220

Leu Ser Ala Leu Leu Ala Gly Lys Leu Ala Leu Glu Ala Gly Ile Pro

225 230 235 240

Pro Gly Val Leu Asn Ile Val Ser Gly Tyr Gly Pro Thr Ala Gly Ala

245 250 255

Ala Ile Ala Glu His Met Asp Val Asp Lys Val Ala Phe Thr Gly Ser

260 265 270

Thr Glu Val Gly Lys Leu Val Met Gly Ala Ala Ala Arg Ser Asn Leu

275 280 285

Lys Pro Val Thr Leu Glu Leu Gly Gly Lys Ser Pro Met Ile Val Cys

290 295 300

Glu Asp Ala Asp Val Asp Ala Ala Val Glu Leu Ala His Phe Ala Ile

305 310 315 320

Phe Phe Asn Gln Gly Gln Cys Cys Cys Ala Gly Ser Arg Thr Phe Val

325 330 335

His Glu Ser Ile Tyr Asp Glu Phe Val Glu Lys Ser Lys Ala Arg Ala

340 345350

Leu Lys Arg Val Val Gly Asp Pro Phe Arg Ser Gly Val Glu Gln Gly

355 360 365

Pro Gln Val Asn Lys Glu Gln Phe Asn Lys Val Leu Ser Tyr Ile Glu

370 375 380

Ser Gly Gln Gln Gln Gly Ala Asn Leu Leu Thr Gly Gly Gly Arg Leu

385 390 395 400

Gly Asn Val Gly Tyr Tyr Ile Lys Pro Thr Ile Phe Thr Asp Val Lys

405 410 415

Asp Gly Met Lys Ile Phe Asp Glu Glu Ile Phe Gly Pro Val Gln Thr

420 425 430

Ile Ala Lys Phe Lys Thr Leu Glu Asp Val Val Gln Arg Ala Asn Asn

435 440 445

Thr Val Tyr Gly Leu Ala Ala Gly Ile Phe Ser Asn Asn Leu Asn Thr

450 455 460

Val Asn Thr Leu Ser Arg Ala Leu Arg Ala Gly Thr Val Trp Val Asn

465 470 475 480

Cys Phe Asp Val Phe Asp Ala Thr Ile Pro Phe Gly Gly Tyr Lys Gln

485 490 495

Ser Gly Ile Gly Arg Glu Lys Gly Lys Glu Ala Leu Asp Asn Tyr Thr

500 505510

Gln Val Lys Ala Val Val Thr Pro Ile His Asn Pro Ala Trp Leu

515 520 525

<210>5

<211>506

<212>PRT

<213> Erythrophora dentata (Syntricia caninervis)

<400>5

Met Gly Leu His Ala Glu Ile Val Pro Gln Arg Arg Leu Phe Ile Asp

1 5 10 15

Gly Asp Trp Val Gln Pro Arg Gln Gly Lys Arg Ile Pro Ile Val Asn

20 25 30

Pro Thr Thr Glu Glu Ser Val Gly Asp Ile Pro Ala Ala Thr Ser Glu

35 40 45

Asp Val Tyr Ala Ala Val Lys Ala Ala Lys Glu Ala Leu His Arg Asn

50 55 60

Asn Gly Lys Asp Trp Ser Lys Ala Thr Gly Lys His Arg Ala Thr Tyr

65 70 75 80

Leu Arg Ala Ile Ala Lys Arg Val Ala Glu Arg Lys Asp Glu Leu Ala

85 90 95

Lys Leu Glu Ser Ile Asp Cys Gly Lys Pro Leu Asp Glu Ala Ala Trp

100 105 110

Asp Met Asp Asp Val Ser Gly Cys Phe Glu Tyr Tyr Ala Asp LeuAla

115 120 125

Glu Lys Leu Asp Glu Arg Gln Tyr Ala Pro Leu Glu Leu Pro Met Glu

130 135 140

Gln Phe Lys Cys Asn Ile Leu Arg Gln Pro Val Gly Val Val Ala Leu

145 150 155 160

Ile Thr Pro Trp Asn Tyr Pro Leu Leu Met Ala Thr Trp Lys Val Ala

165 170 175

Pro Ala Leu Ala Ala Gly Cys Thr Ala Ile Leu Lys Pro Ser Glu Ile

180 185 190

Ala Ser Val Thr Cys Leu Glu Leu Ala Ser Ile Ala Lys Glu Val Gly

195 200 205

Leu Pro Asn Gly Val Leu Asn Val Ile Thr Gly Tyr Gly Gln Glu Ala

210 215 220

Gly Ala Pro Leu Ala Ser His Gln Asp Val Asn Lys Val Ala Phe Thr

225 230 235 240

Gly Ser Thr Asp Thr Gly Arg Ser Ile Met Ser Ala Ala Ser Gln Leu

245 250 255

Ile Lys Pro Val Thr Leu Glu Leu Gly Gly Lys Ser Pro Ile Ile Val

260 265 270

Phe Glu Asp Ala Asp Ile Asp Lys Ala Val Glu Trp Ala Met Phe Gly

275 280 285

Ala Phe Trp Thr Asn Gly Gln Ile Cys Ser Ala Thr Ser Arg Leu Leu

290 295 300

Leu Gln Glu Ser Ile Ala Asp Glu Phe Leu Lys Lys Ile Ala Ser Trp

305 310 315 320

Ala Ser Ser Ile Lys Val Ser Asp Pro Leu Glu Lys Asp Cys Arg Leu

325 330 335

Gly Pro Leu Val Ser Glu Asn Gln Tyr Lys Lys Val Lys Glu Phe Val

340 345 350

Arg Val Ala Gln Glu Glu Gly Ala Thr Leu Val Cys Gly Gly Lys Arg

355 360 365

Pro Asp His Leu Ser Lys Gly Tyr Phe Leu Ala Pro Thr Val Leu Ser

370 375 380

Asn Val Lys Arg Asp Met Gln Ile Trp Ala Glu Glu Val Phe Gly Pro

385 390 395 400

Val Leu Ala Val Ser Thr Phe Lys Thr Glu Glu Glu Ala Val Leu Met

405 410 415

Ala Asn Asp Thr Gln Tyr Gly Leu Gly Gly Ala Val Ile Ser Lys Asp

420 425 430

Asp Glu Arg Cys Lys Arg Val Ser Glu Ala Leu Glu Cys Gly Ile Val

435 440 445

Trp Ile Asn Cys Ser Gln Pro Cys Phe Cys Gln Ala Pro Trp Gly Gly

450 455 460

Asn Lys Arg Ser Gly Phe Gly Arg Glu Leu Gly Glu Trp Gly Leu Glu

465 470 475 480

Asn Tyr Leu Thr Val Lys Gln Ile Thr Arg Tyr Ile Ser Asn Asp Asp

485 490 495

Trp Gly Trp Tyr Pro Lys Pro Ser Lys Leu

500 505

<210>6

<211>496

<212>PRT

<213> Erythrophora dentata (Syntricia caninervis)

<400>6

Met Ala Gly Gln Gly Phe Phe Gln Asp Ile Leu Asp Gly Asp Val Phe

1 5 10 15

Lys Phe Tyr Ala Asp Gly Glu Trp Lys Thr Ser Thr Ser Gly Lys Ser

20 25 30

Val Gly Ile Thr Asn Pro Ser Thr Leu Lys Thr Ala Phe Lys Val Gln

35 40 45

Ala Cys Thr Gln Asp Glu Val Asn Lys Ala Ile Glu Ser Ala Lys Val

50 55 60

Ala Gln Lys Ala Trp Ala Lys Thr Pro Leu Trp Lys Arg Ala Glu Ala

65 70 75 80

Leu His Arg Phe Ala Gly Ile Leu Lys Asp Gln Lys Asn Val Ile Ala

85 90 95

Glu Cys Leu Val Lys Glu Val Ala Lys Ala Gln Lys Asp Ser Val Thr

100 105 110

Glu Val Val Arg Ser Gly Asp Leu Ile Ser Tyr Ser Ala Glu Glu Gly

115 120 125

Ile Arg Ile Met Ala Glu Gly Lys Phe Leu Ala Ser Asp Ser Phe Pro

130 135 140

Gly Asn Gly Arg Asn Lys Tyr Cys Leu Ala Ser Lys Thr Pro Leu Gly

145 150 155 160

Val Val Leu Cys Ile Pro Pro Phe Asn Tyr Pro Val Asn Leu Ala Val

165 170 175

Ser Lys Ile Ala Pro Ala Leu Ile Thr Gly Asn Ala Val Ile Leu Lys

180 185 190

Pro Pro Thr Gln Gly Ala Val Ser Ala Leu His Met Val His Cys Ala

195 200 205

His Met Ala Gly Phe Pro Lys Gly Leu Ile Ala Ala Val Thr Gly Lys

210 215 220

Gly Ser Glu Ile Gly Asp Leu Leu ThrMet His Pro Gly Ile Asp Cys

225 230 235 240

Ile Ser Phe Thr Gly Gly Asp Thr Gly Ile Ala Ile Ser Lys Lys Ala

245 250 255

Gly Met Ile Pro Leu Gln Met Glu Leu Gly Gly Lys Asp Cys Cys Ile

260 265 270

Val Leu Glu Asp Ala Asp Leu Glu Leu Ala Ala Asn Asn Val Ile Lys

275 280 285

Gly Gly Tyr Ser Tyr Ser Gly Gln Arg Cys Thr Ala Ile Lys Val Ile

290 295 300

Cys Val Met Glu Ser Val Ala Glu Glu Leu Val Lys Asn Ile Val Asp

305 310 315 320

Lys Met Ala Lys Leu Lys Val Gly Met Pro Glu Asp Asn Cys Asp Ile

325 330 335

Thr Pro Val Val Ser Gln Ser Ser Ala Asn Tyr Ile Gln Gly Leu Val

340 345 350

Glu Asp Ala Gln Ala Lys Gly Ala Lys Phe His Gln Glu Trp Lys Arg

355 360 365

Glu Asn Asn Leu Ile His Pro Leu Leu Ile Asp Asn Val Thr Pro Asp

370 375 380

Met Arg Ile Ala Trp Glu Glu Pro Phe Gly ProVal Ile Pro Val Ile

385 390 395 400

Arg Ile Lys Thr Val Glu Glu Gly Ile His His Cys Asn Ala Asn Asn

405 410 415

Phe Ala Leu Gln Gly Cys Val Phe Thr Lys Asp Ile Asn Lys Ala Ile

420 425 430

Leu Val Ser Asn Ala Met Glu Ser Gly Thr Ile Gln Ile Asn Ala Ala

435 440 445

Pro Ala Arg Gly Pro Asp His Phe Pro Phe Gln Gly Leu Arg Asp Ser

450 455 460

Gly Ile Gly Ser Gln Gly Val Thr Asn Ser Ile Leu Met Met Thr Lys

465 470 475 480

Val Lys Ser Thr Val Ile Asn Leu Pro Val Glu Ser Tyr Thr Met Gly

485 490 495

Claims

1. An extremely dry-tolerant erythroaldehyde dehydrogenase gene, which is characterized in that the genes are named asScALDH2B2、ScALDH10A1AndScALDH11A1；

the aldehyde dehydrogenase geneScALDH2B2The length of the coding region is 1584bp, the nucleotide sequence is SEQ ID NO.1, 527 amino acids are coded, and the amino acid sequence is SEQ ID NO. 4;

the aldehyde dehydrogenase geneScALDH10A1The length of the coding region is 1521bp, the nucleotide sequence of the coding region is SEQ ID NO.2, 506 amino acids are coded, and the amino acid sequence of the coding region is SEQ ID NO. 5;

the aldehyde dehydrogenase geneScALDH11A1The length of the coding region is 1491bp, the nucleotide sequence is SEQ ID NO.3, 496 amino acids are coded, and the amino acid sequence is SEQ ID NO. 6.

2.The extremely dry-tolerant erythroaldehyde dehydrogenase gene prokaryotic system function test body as claimed in claim 1, characterized in that the nucleotide sequences SEQ ID No.1-3 are respectively subjected to prokaryotic expression vector construction according to a conventional method, and are transduced to DE3 strain of prokaryotic cell Escherichia coli BL21 to respectively obtain three types of trans-genes with stress resistanceScALDH2B2、 ScALDH10A1AndScALDH11A1the prokaryotic transformant of (1).

3. Use of the extreme dry-tolerant erythroaldehyde dehydrogenase gene according to claim 1 for the preparation of arabidopsis thaliana breeding for enhanced resistance to drought stress and salt stress.