CN114854779A - Tomato ascorbic acid biosynthesis gene PMI and application thereof - Google Patents

Tomato ascorbic acid biosynthesis gene PMI and application thereof Download PDF

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CN114854779A
CN114854779A CN202210403837.5A CN202210403837A CN114854779A CN 114854779 A CN114854779 A CN 114854779A CN 202210403837 A CN202210403837 A CN 202210403837A CN 114854779 A CN114854779 A CN 114854779A
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ascorbic acid
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张余洋
叶志彪
郑伟
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Wuhan Chuwei Biotechnology Co ltd
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Abstract

The invention belongs to the technical field of genetic engineering, and discloses a tomato ascorbic acid biosynthesis gene PMI and application thereof, wherein the tomato ascorbic acid biosynthesis gene PMI is PMI1 and PMI2, and the DNAs of ORFs of PMI1 and PMI2 are SEQ ID NO: 1 and SEQ ID NO: 2; the protein encoded by the gene PMI for the biosynthesis of ascorbic acid in tomato comprises the amino acid sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4. the invention takes the conventional tomato strain AC as an object, clones key genes of a Smirnoff path synthesized by ascorbic acid, constructs a corresponding genetic transformation vector, and transfers the genetic transformation vector into the tomato strain AC by utilizing an agrobacterium-mediated genetic transformation method to ensure that a target gene is over-expressed or inhibited; the obtained transgenic tomato is subjected to ascorbic acid content analysis, and the effect of the genes in controlling the content of the tomato ascorbic acid is evaluated.

Description

Tomato ascorbic acid biosynthesis gene PMI and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a tomato ascorbic acid biosynthesis gene PMI and application thereof.
Background
Currently, the closest prior art:
ascorbic acid (AsA), also known as vitamin c (vc), has important functions in the body, such as antioxidation, cofactors of important enzymes, etc. Researches in plants find that the AsA is positively correlated with the stress resistance of the plants, the content of endogenous AsA is increased, and the stress resistance of the plants, such as cold resistance and the like, can be improved. Meanwhile, ascorbic acid is an important substance for maintaining growth and development of human beings and animals. Whereas primates (e.g. humans) lack the last step in the AsA biosynthetic pathway and therefore cannot synthesize AsA, only sufficient quantities of AsA can be obtained from plants (Jain et al, 2000), with fresh fruit being the main source of ascorbic acid.
Although ascorbic acid has a very important role in the nutrition required in humans, the understanding of its biosynthetic pathway is not completely complete and the research of the plant ascorbic acid synthetic pathway has started in the late animal, until 1998 the plant ascorbic acid Smirnoff synthetic pathway was first proposed. Subsequent studies have found that ascorbic acid synthesis in plants is different from that in animals and that there may be multiple synthetic pathways, and thus plant ascorbic acid synthesis is much more complex than in animals.
Despite the important functions of ascorbic acid, its molecular structure has been identified in the last 30 centuries. With the development of scientific technology, differences in ascorbic acid synthesis pathways of animals, microorganisms and plants have been found. Biosynthetic pathways for animals and microorganisms were revealed earlier. Lsherwood et al proposed in 1954 that an ascorbic acid synthesis pathway similar to that of animals exists in plants, but no intermediate was detected and thus not validated. By the 90 s, ascorbic acid-deficient mutants of Arabidopsis thaliana were isolated by the predecessors, and this has led to considerable and significant progress in the plant ascorbic acid biosynthetic pathway. In 1998, Wheeler et al (Wheeler et al, 1998) proposed the first ascorbic acid pathway, D-mannose/L-galactose, by isotopic tracing, biochemical analysis and early research results. Since then, three pathways have been proposed in succession, namely the galacturonic acid pathway, the gulose pathway and the inositol pathway.
Currently, no research on aspects related to PMI transgenes has been reported. Two PMIs with higher homology exist in the tomato genome and are named as PMI1 and PMI2 respectively. The overexpression and co-suppression expression vectors of PMI1 and PMI2 are constructed, and are introduced into a tomato line AC by a genetic transformation method by utilizing an agrobacterium-mediated method, so that a target gene is overexpressed or suppressed. And (3) measuring the ascorbic acid content of leaves and fruits of the transgenic tomatoes, and preliminarily identifying the functions of the two PMIs in an ascorbic acid synthesis pathway through analysis of experimental results.
Tomatoes are one of the vegetable crops that are commonly cultivated all over the world and occupy an important position in the development of the vegetable industry. Tomatoes are rich in ascorbic acid and are an important source of dietary vitamins. Tomatoes are widely studied as a model plant because of their small genome, short growth cycle, ability to be grown annually, mature genetic transformation systems, and self-pollinating plants. Although some progress has been made in the study of the ascorbic acid metabolism of plants at present, most of them have focused on the model plant Arabidopsis thaliana. In tomato, only limited genes related to ascorbic acid metabolism have been cloned and identified, and studies on the mechanism regulating ascorbic acid accumulation in tomato are only in the initial stage.
The content of target products in transgenic plants or the synthesis of exogenous metabolites can be improved by improving the activity of a rate-limiting enzyme for controlling the synthesis of a specific metabolite through genetic engineering or introducing a new metabolite synthesis path in plants. The development of various important agronomic traits in crops by means of genetic improvement has been a trend in the future. Therefore, in order to more effectively increase the level of ascorbic acid in tomato, the cloning and functional identification of genes encoding enzymes involved in the metabolic pathway of ascorbic acid is a key task.
In summary, the problems of the prior art are as follows:
(1) only a few ascorbic acid anabolic genes have been cloned and identified in tomato, and there has been little research on the mechanism regulating ascorbic acid accumulation in tomato. In order to accelerate the genetic improvement of the ascorbic acid content in tomato more effectively, the functional identification of the relevant genes PMI1 and PMI2 in the ascorbic acid metabolic pathway is a key task.
(2) At present, concrete experimental evidence of how PMIs genes play a role in regulation of tomato ascorbic acid synthesis and metabolism is lacking. Therefore, the invention leads the PMI1 and PMI2 genes to be over-expressed in the tomato, systematically measures the expression quantity of corresponding genes of transgenic materials and the content of ascorbic acid, and discloses the specific functions of PMI1 and PMI2 genes in the tomato. … …
The difficulty of solving the technical problems is as follows:
since ascorbic acid anabolism is a quantitative trait, the major gene has not been elucidated, and genetic improvement of the ascorbic acid content in tomato is limited. Functional analysis of related genes is required by molecular biology to verify the role of PMIs genes in ascorbic acid synthesis.
The significance of solving the technical problems is as follows:
tomato is an important vegetable crop, the content of ascorbic acid is one of important traits for measuring the quality of tomato fruits, and the precursor condition for improving the trait is that the gene functions in the ascorbic acid synthesis and redox pathways and the regulation mechanism of the ascorbic acid synthesis and redox pathways are required to be comprehensively understood. At present, the regulation mechanism of genes related to ascorbic acid biosynthesis is less reported. The invention mainly discloses the effect of PMI genes in the synthesis of ascorbic acid in tomatoes, and the effect and the regulation mechanism of the PMI genes in the regulation of the biosynthesis of the ascorbic acid are researched by carrying out excessive or suppressed expression on the PMI1 and the PMI2 in the tomatoes. This will help to increase the level of ascorbic acid in tomato by genetic improvement method, and provide new theoretical technique and support for tomato variety improvement and cultivation.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a tomato ascorbic acid biosynthesis gene PMI and application thereof. The invention takes a tomato (tomato lycopersicum) conventional strain AC (stored in a laboratory) as an object, clones ascorbic acid to synthesize a key gene of a Smirnoff pathway, constructs a corresponding sense and RNAi genetic transformation vector, and transfers the vector into the tomato strain AC by utilizing an agrobacterium-mediated genetic transformation method to ensure that a target gene is over-expressed or inhibited to express. The obtained transgenic tomato is subjected to ascorbic acid content analysis to evaluate the function of the genes in controlling the content of the ascorbic acid in the tomato.
The invention is realized by the tomato ascorbic acid biosynthesis gene PMI which is PMI1 and PMI2, and DNAs of ORFs of PMI1 and PMI2 are respectively (Solyc02g086090.2) CTCCCATCGTCACTTGCTTTGTCCAAATCCCCAACTTCCCCTCCTCCTCACA ACACTTTGCCCCTTCAAAAACTAACCCCCCAATGGAAACTATTCAATTCTC TTCAACTTCAACTTCTTTGTTTATTTCAACTTCTTTCTTTTTCTCTCGGGTCT CTCAACAACAATGGAGATGGAGGAAGGTTTTAAGGGGTTACTCAGGTTAA TTGGTTCTGTTAAGAATTACGATTGGGGCCGTACGGCGAAGGAGTCTTGTG TTGGACGTCTGTATAGGCTCAATTCTCGGACGAAAATTGATGAAAAACAGC CATATGCCGAGTTTTGGATGGGAACTCATGACTCTGGACCATCCTACATTGT GGTGGAACGAGGAGGAAGAATTCAGAATGGACATGCTAATGGCGGAGGA ATTAGAGACAAGTGTTCTTTGAAAGATTGGATTCAGAAGAACCCTAGTGTA CTTGGAGAAACTGTTCTTGCCAAATGGGGTACCCAGCTTCCTTTTCTCTTC AAGGTACTCTCAATTGAGAAAGCTTTGTCTATACAAGCTCATCCAGACAAG GATCTGGCAATTCTTTTGCATAAGGAGCAGCCACTCGTTTACAAGGATGAT AACCACAAACCTGAGATGGCTTTGGCATTGACCAAGTTCGAGGCCTTGTG TGGCTTCATAAGTCTTGAGGAGCTTAAAGTGATTGTTCAGACTGTGCCCGA GATTGTTGAAGTAGTTGGTAATGCGCTTGCAGAGCTAGTATTGGACTTGAG CGAGGATGATGAAGAGGAGAAAGGTAAATTAGTGCTCAGAAAATTATTCA CGGAGATTATGTCAGCTAGCAAGGATGTGATCACGGAAGTACTTGCTAAGC TGATTAGTCGTCTGAACATTAAAAACAAGGTAAGGGTGCTGACCGACAAG GAACAACTGGTCCTAGGACTTGAGAAGCAGTATCCATCTGATGTTGGTGTT TTAGCAGCATTCTTGTTTAATTACGTGAAGCTCAATCCTGGTGAAGCTTTAT ATTTAGGGGCAAATGAACCCCATGCATATGTATATGGAGAATGTGTCGAATG TATGGCAACCTCAGATAATGTGGTACGCGCTGGCCTAACTCCCAAGCACCG GGATGTTAGAACTCTCTGTTCAATGCTCACGTATAGACAGGGTAACCCTGA AATTCTGCACGGTACGGCAATAAATCCATACACAGTGAGATACCTCCCTCC TTTTGATGAATTCGAGGTGGATCATTGCATTCTCCCCCCATATTCAACTGTT ACCTTCCCTTCTGCTCCTGGTCCGTCCATGTTTTTGGTCATGGGAGGAGAG GGAACAATGACCACATCAGCAGAAGTGATTGTGGTTGAAGGTGATGTCCT ATTTGCACCTGCAAACACCAATATTACCATTGCAACCTCCTCTGGTTTGCAC TTGTATAGAGCAGGTGTAAACAGCAGATTTTTTGAGGAATGATAGTTGTAG GCTTGTAGCCCCTTATGCTTGCTAATAAAACAGTGAATTTTTCTGTTACACT GGCTGTTCCACCTTGTATTAATAGCATGTTCAGGTATATAACCGCTTAAGTTA AGTGTA
And (Solyc02g063220.2) TAGGAAAGGAAGAAATATTTAAAGTTGGCAGGAAAATGTTTTCCCTTAATT AAATTTGAAACCGCTGCTTTGCGTGTGATAAAGAAAAAAACACCAAAACT TGTAAATCCTCACTAATTTTTCTTGAATGGTACAACACCAACATTCATGTGC TCCTCTGTCAATCTTCTTCTTCTTCAAATCATCATCATCTTTGTTTGAACTGA AGCTACTGTTTCCCGTTCCTTGCTCTGAAGCATGAACCAAGACTACTAAAC CTTTCGCAGAGGAAAAAAACACGAGAGCTTTTTTTTTTTAAACTTAAAATC TGTTTTATGGACACTGACTTGTTGTCGGTGACGGAAGGGAGAGGGAGGCT GGTGAGGTTGATGGGTTGCGTGAAGAATTACGATTGGGGACCACCCGGGA AGGAATCTCGTGTAGCGCGGTTGTATGCTTGCAATAGTGGTAACTATGTTG ACCAAGAGAAGCCTTATGCCGAATTTTGGATGGGTACTCACGATTCTGGGC CTTCCTATGTTGTGGAAGGAACTGAGAATGGGTTGGTTAATGGTAAAGGAG AGGGACACAAGTTAACATTGAAGAATTGGATTCAAAACAACCCTAATGTTC TTGGAGAGAAGGTTGTGAAGAAATGGGGTACCAACCTTCCTTTTCTCTTCA AGGTACTATCTGTTGCAAAAGCTTTGTCCATACAGGCCCATCCAGACAAGG ATTTAGCCTCTCGTCTGCATAGTGAGCTTCCGGATGTTTATAAGGATGACAA TCACAAACCGGAGATGGCATTGGCGTTGACGGAATTTGAGGCATTGTGTG GATTTATAAGTCTCGAGGAGCTTAAGTTGATTGTTCAAACTGTGCCAGAGA TTGTTGAATTGGTCGGTACAGCACACACAGAGCAGGTATTGGAATTGAAC GAGGATGATGGGAAGGAAAAAGGTAAATTCGTCCTACAATCAGTATTTACT GAGCTGATGTCAGCAAACAAGGATGTGGTTGCTGAAGTGATAGCCAAGCT GATTAGTCGCCTACACGTTAAAAATCAGGCAAGGGAGCTGACAGAGAAAG AACAAGTGGTGCTTAGACTTGAGAAGCAGTATCCAGCTGATATTGGTGTCT TGGCTGCATTCTTGTTAAATTATGTGAAACTCAATCCTGGTGAAGCCTTATA TTTAGGAGCAAATGAACCTCATGCTTATTTATATGGTGATTGTATTGAATGCA TGGCAACATCGGACAACGTTGTTCGCGCTGGCCTAACTCCAAAACACCGA GATGTTAAAACACTATGCTCAATGCTCACTTACAGACAGGGTTTTCCTGAA ATTCTGCAGGGCACCGCAGTAAATCCTCATGTTATGAGGTACATTCCTCCTT TTGATGAATTTGAAGTTGATCGTTGTATTCTTCCCGAACAATCAACTACTGA ATTTCCATCTATTCCCGGTCCATCCATTTTTATGGTCGTGGAGGGAGAGGGA ACATTAACCTCATCATCAGACGAGATTATCCATGAAGGTGATGTCCTTTTTG CACCTGCAAACACCAACATTACTGTCTCGACATCTTCTGGTTTGCAATTATA TAGAACAGGAATAAACAGCAGGTTTTTTGAGGAGTGAAGGTTGTATACGTA TTCTAGTACATAAAATTGTTCATTAATTTTTCTCTATAGGAAAATCGGCTGAT CCAACCTTGTAATATTAGCCATTTCTGTAAATCAGGTATACAAATAAATATGA CTTGTTATAGTTGCCTCATTGTACTAGTTCAGTGTTTCACAATCTAAGTGCA ATAGGTGGAATTAGTATAGGGATTAGGATTTAAGGTTTTGTAAGATACTTTC AAATGCTTTTAATCCAAAACAAAAAGAGCACAGGTATAATTCCCAACAAA AGAAAAAGAGATTGGAGCAGACAATACAGTAATCTTGTGTGCTGTCAAC; the Solyc02g086090.2 fragment is 1591 bp; the Solyc02g063220.2 fragment is 1945 bp; the consensus at the PMI1 and PMI2 nucleic acid level was 79%.
Another object of the present invention is to provide a protein encoded by PMI, which comprises (PMI1 enzyme protein) MEMEEGFKGLLRLIGSVKNYDWGRTAKESCVGRLYRLNSRTKIDEKQPY AEFWMGTHDSGPSYIVVERGGRIQNGHANGGGIRDKCSLKDWIQKNPSVLG ETVLAKWGTQLPFLFKVLSIEKALSIQAHPDKDLAILLHKEQPLVYKDDNHKP EMALALTKFEALCGFISLEELKVIVQTVPEIVEVVGNALAELVLDLSEDDEEE KGKLVLRKLFTEIMSASKDVITEVLAKLISRLNIKNKVRVLTDKEQLVLGLEK QYPSDVGVLAAFLFNYVKLNPGEALYLGANEPHAYVYGECVECMATSDNVV RAGLTPKHRDVRTLCSMLTYRQGNPEILHGTAINPYTVRYLPPFDEFEVDHCIL PPYSTVTFPSAPGPSMFLVMGGEGTMTTSAEVIVVEGDVLFAPANTNITIATSS GLHLYRAGVNSRFFEE ×, respectively, using said tomato ascorbic acid biosynthesis gene
And (PM2 enzyme protein) MDTDLLSVTEGRGRLVRLMGCVKNYDWGPPGKESRVARLYACNSGNYVDQ EKPYAEFWMGTHDSGPSYVVEGTENGLVNGKGEGHKLTLKNWIQNNPNVL GEKVVKKWGTNLPFLFKVLSVAKALSIQAHPDKDLASRLHSELPDVYKDDN HKPEMALALTEFEALCGFISLEELKLIVQTVPEIVELVGTAHTEQVLELNEDDG KEKGKFVLQSVFTELMSANKDVVAEVIAKLISRLHVKNQARELTEKEQVVLR LEKQYPADIGVLAAFLLNYVKLNPGEALYLGANEPHAYLYGDCIECMATSDN VVRAGLTPKHRDVKTLCSMLTYRQGFPEILQGTAVNPHVMRYIPPFDEFEVDR CILPEQSTTEFPSIPGPSIFMVVEGEGTLTSSSDEIIHEGDVLFAPANTNITVSTSS GLQLYRTGINSRFFEE;
the PMI1 enzyme protein consists of 434 amino acids; the PMI2 enzyme protein consists of 435 amino acids; the similarity at the amino acid level of the PMI1 enzyme protein and the PMI2 enzyme protein was 70%.
Another objective of the invention is to provide an expression vector pMV2 (detailed vector diagram in FIG. 7) constructed by using the tomato ascorbic acid biosynthesis gene PMI.
Another object of the present invention is to provide a method for constructing the expression vector, which comprises:
firstly, designing a full-length gene Primer by using Primer5, and amplifying in a PCR (polymerase chain reaction) instrument by using cDNA (complementary deoxyribonucleic acid) of tomato AC (alternating current) as a template to obtain a target fragment; linking the PCR product to a pEASY-B (pEASY-B is a public commercial vector, detailed vector information is shown in Beijing all-open gold biotechnology limited (www.transgen.com.cn)) vector, connecting the product to transform escherichia coli by a heat shock method, screening positive clones by a 50mg/L Km resistance plate, selecting the positive clones, carrying out shake culture on a shaker at 37 ℃ for overnight at 200r/min, carrying out PCR detection by using a gene specific primer, and then selecting the positive clones for sequencing verification;
the sequence of pEASY-B is:
AGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGG AAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCC GGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTGC CCTTAAGGGCAGCTTCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGG AAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGC ACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGT GTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCT CGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACC TCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACG TTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGA TTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACA AAATTCAGGGCGCAAGGGCTGCTAAAGGAAGCGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGA ATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGG CGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAA GCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGACAG GATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGC TATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTT TGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCG TTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGAT CTCCTGTCATCCCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCC GGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATC AGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGC GAGGATCTCGTCGTGACCCACGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGA CTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCG AATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGAC GAGTTCTTCTGAATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTT TGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTA CATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTA AAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAG AATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGC CATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGC ACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGAC ACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACA ATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTG ATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTA GTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAA GCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCT AGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTA GAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACC AGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAA ATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTA ATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAA GGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACC TACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGA ACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACT TGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCC TGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTT GAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAG
secondly, selecting the clone shake bacteria with correct sequence comparison to extract plasmids; sequences as described previously for gene accession no: solyc02g086090.2 and Solyc02g063220.2. Xbal and KpnI double enzyme digestion recombinant plasmid for 1.5 h;
thirdly, cutting the gel, recovering the gel by using a gel recovery kit to obtain a target fragment, and connecting the target fragment to a pMV2 vector subjected to double enzyme digestion by Xbal and Kpn I by using T4 ligase; connecting the products, transforming escherichia coli by a heat shock method, selecting positive clones, carrying out PCR positive detection on the single clones by using a 35S binding gene reverse specific primer, selecting the positive clones, shaking the bacteria to extract plasmids, carrying out double enzyme digestion verification, and transferring the plasmids into agrobacterium-induced C58 cells by an electric shock method after the verification is correct;
fourthly, selecting positive clones, carrying out shake culture at 150r/min on a shaking table at 28 ℃ overnight, and carrying out PCR positive detection on the bacterial liquid by using 35S plus gene reverse specific primers on the carrier; adding glycerol into the positive clone, mixing uniformly, and storing in a low-temperature refrigerator at-70 ℃.
Further, in the first step, the PCR amplification method comprises:
a10-microliter reaction system is adopted, EF1a is used as an internal reference, and the assay is carried out by a Roche fluorescence quantitative PCR instrument LC480, and the system comprises
Figure RE-GDA0003729433390000091
Premix Ex Taq TM (2X) 5. mu.L of each forward and reverse primer, 4. mu.L of template;
each sample was replicated three times, the reaction procedure: pre-denaturation at 95 ℃ for 5 min; the specificity of PMI1 and PMI2 genes was determined by melting curve analysis after 45 cycles at 95 ℃ for 10s and 58 ℃ for 1 min.
The invention also aims to provide an application of the tomato ascorbic acid biosynthesis gene PMI in measurement of the relative expression quantity of leaves of PMIs over-transgenic plants and the content of ascorbic acid, and the application method of PMIs over-transgenic plants in measurement of the relative expression quantity of leaves and the content of ascorbic acid comprises the following steps:
screening out the excessive transgenic plants with relatively high expression quantity of the T0 generation, and detecting and confirming the T1 generation again; taking 3 ultra-transgenic lines and non-transgenic lines as materials, and determining and analyzing relative expression quantity by qPCR;
the content of ascorbic acid is measured by an enzyme-linked immunosorbent assay (ELISA) instrument by adopting a method of HCl extraction; quickly freezing the transformed plant young and tender leaves with liquid nitrogen, grinding the sample in the liquid nitrogen into powder, subpackaging 0.1g of the sample in a 2ml centrifuge tube, and repeating each sample for 3 times;
during measurement, 1ml of precooled 0.2mol/L HCl solution is added into each tube for extraction, about half an hour of extraction is carried out, the mixture is inverted and uniformly mixed every 5min, and the mixture is centrifuged at 12000r/min at 4 ℃ for 10 min;
adding 50 μ L of 0.2mol/L NaH2PO4(pH 5.6) into 500 μ L of the supernatant, and adjusting the pH value to 5 and 6 with 0.2mol/L NaOH;
after mixing evenly, 100 mu L of supernatant is taken and added with 140 mu L of 0.12mol/L NaH in turn 2 PO 4 And 10. mu.L of 25mmol/L DTT, and reacted at room temperature for 30min in the dark. Taking 95 mu L of supernatant, adding 0.1ml of 0.2mol/L NaH 2 PO 4 Measuring the value of the absorption wavelength at 265nm by using an enzyme-labeling instrument;
finally, 5 mu L of 40U/ml AO enzyme is added, and the absorption wavelength value is measured after the reaction; the standard ascorbic acid solution is measured by a microplate reader, and a standard curve is drawn.
In summary, the advantages and positive effects of the invention are:
the invention clones ascorbic acid synthesis related genes from tomatoes, over-expresses the ascorbic acid synthesis related genes in the tomatoes, systematically measures the corresponding expression amount and the ascorbic acid content of corresponding transgenic materials, and preliminarily identifies the effect of PMIs in the synthesis and metabolic regulation of the ascorbic acid in the tomatoes.
Compared with the prior art, the invention has the advantages that:
1) two members of the mannose-6-phosphate isomerase gene family, PMI1 and PMI2, were cloned, with ORFs 1591bp and 1945bp, respectively, encoding 434 and 435 amino acids, respectively, with 79% identity at the nucleic acid level and 70% similarity at the amino acid level.
2) The results of tissue expression profiling analysis of PMI1 and PMI2 show that PMIs are constitutively expressed in each tissue of tomato, but the expression level of PMIs in each organ is greatly different. PMI1 was expressed in the highest amount in leaves, followed by roots and flowers, and in the lowest amount in stems and ripe fruit; PMI2 was expressed in the highest amount in flowers, followed by leaves, and showed a tendency to decrease and then increase at different stages of the fruit.
3) The method determines the content of the ascorbic acid in roots, stems, leaves, flowers and fruits in each development period of the tomato line AC, and the result shows that the total ascorbic acid content has great difference, the content in the roots is the lowest, and the content in the leaves is the highest; the total ascorbic acid content showed a tendency to increase gradually as the fruit developed, with the highest ascorbic acid content in the red ripe fruit.
4) The invention constructs PMI1 and PMI2 overexpression vectors, and transfers the overexpression vectors into the conventional tomato strain AC by utilizing an agrobacterium-mediated genetic transformation method. The obtained tomato transformation plant is detected by PCR, and the result shows that the exogenous gene is inserted into the tomato genome.
5) The expression quantity of the target gene in the blade of the excessive transgenic strain is obviously higher than that of the non-transgenic material, and the expression quantities of the PMI1 excessive transgenic strains O2-6, O11-7 and O12-2 are 157.7 times, 338.9 times and 156.4 times of that of the non-transgenic material respectively; the expression amounts of the PMI2 excessive transgenic strains O4-2, O12-11 and O18-9 are 11.6 times, 33.3 times and 20.3 times of the non-transgenic material respectively. The fold of the overexpression of PMI1 was higher than the fold of the overexpression of PMI2, which is likely due to the lower background expression of PMI1 in the leaves than PMI 2.
6) The content of total ascorbic acid in the leaves of PMI1 and PMI2 excessive transgenic lines is measured by an enzyme-labeling instrument, the content of the total ascorbic acid in the leaves of a tomato is remarkably improved by PMI1 excessive expression, the content of the total ascorbic acid in the leaves of the tomato is also improved by PMI2 excessive expression, after PMI1 is up-regulated and expressed, the up-regulated expression of APX1 is promoted, the expression of GalUR, GalDH and MIOX is inhibited, and the expression of most genes in the ascorbic acid synthesis step is not influenced; expression of ascorbic acid anabolism related genes GLDH, GMP, MDHAR, GME1, GME2, DHAR, APX1, GGP, APX6, PMI1, PMI2, GR, GalUR, GalDH and MIOX is promoted after PMI2 is up-regulated, wherein the expression amount of GGP is 5.0 times of that of non-transgenic, but the expression amounts of GME1 and GME2 are obviously inhibited and are respectively reduced to 37.0% and 16.1% of that of non-transgenic.
Drawings
Fig. 1 is a technical route diagram provided by an embodiment of the present invention.
FIG. 2 is a graph showing the tissue expression profiles of PMIs in different tissues of tomato and the total ascorbic acid content in different tissues of tomato according to the present invention.
FIG. 3 is a PCR assay of transgenic plants provided in the examples of the invention.
In the figure: m: labeling with molecular weight; n: negative control; p: positive control (recombinant plasmid); a:1 to 5 are PMI1 transgenic plants with excessive weight; b, 1 to 15 are PMI2 excessive transgenic plants; c, 1 to 14 are PMIs co-suppression transgenic plants; the primers used for PCR detection were 35S plus gene reverse primers.
FIG. 4 is a diagram showing the analysis of the expression of a target gene in leaves of a transgenic line according to the present invention.
In the figure: o2, O11, O12, O17 and O18 are PMI1 excess strains; o4, O5, O6, O12, O14, O16, O17, O18, O22 and O26 are PMI2 excess strains; WT wild type.
FIG. 5 shows the detection of the expression level of fruits of transgenic lines overexpressing PMI1 provided by the embodiment of the present invention (A, C); measurement of AsA content in fruit of transgenic line overexpressing PMI2 (B, D).
In the figure: WT wild type; o2-6, O11-7 and O12-2 are PMI1 over-plants; o4-2, O12-11 and O18-9 are PMI2 over-plants.
FIG. 6 is a graph showing the expression analysis of genes involved in AsA synthesis in leaves of PMIs over-transgenic lines according to the present invention.
In the figure: WT wild type; o12-2: PMI1 superplants; o4-2: PMI2 superplants.
FIG. 7 is a map of the pMV2 vector provided by the example of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Ascorbic acid (AsA) is an antioxidant and plays an important role in a number of physiological processes in plants. Ascorbic acid is an important vitamin widely present in fresh fruits and vegetables and in many organisms, and as a biologically active substance it is involved in many metabolic activities. The research on the biosynthesis pathway of the ascorbic acid and the gene engineering research of the metabolic regulation of the ascorbic acid makes a breakthrough. However, only a few ascorbic acid anabolic genes have been cloned and identified in tomato, and regulation of ascorbic acid anabolic in tomato by cloning key genes remains to be advanced. In the prior art, ascorbic acid synthesis related genes are not cloned from tomatoes, and are overexpressed in the tomatoes, corresponding transgenic materials are systematically subjected to measurement of corresponding expression quantity and ascorbic acid content, and a specific theoretical basis for preliminary identification of the effect of PMIs in tomato ascorbic acid synthesis and metabolic regulation is lacked.
Aiming at the problems in the prior art, the invention provides a tomato ascorbic acid biosynthesis gene PMI and application thereof, and the invention is described in detail with reference to the accompanying drawings.
The tomato ascorbic acid biosynthesis gene PMI provided by the embodiment of the invention is PMI1 and PMI2, and the DNAs of the ORFs of PMI1 and PMI2 are SEQ ID NO: 1 and SEQ ID NO: 2; the nucleotide sequence of SEQ ID NO: the 1 fragment is 1591 bp; the nucleotide sequence of SEQ ID NO: the 2 fragment is 1945 bp; the consensus at the PMI1 and PMI2 nucleic acid level was 79%.
The invention provides a protein encoded by a PMI gene for ascorbic acid biosynthesis of tomato, which comprises the following components in percentage by weight of SEQ ID NO: 3 and SEQ ID NO: 4; the nucleotide sequence of SEQ ID NO: 3 is represented by SEQ ID NO: 1 encodes 434 amino acids. The nucleotide sequence of SEQ ID NO: 4 is represented by SEQ ID NO: 2 codes for 435 amino acids; SEQ ID NO: 3 and SEQ ID NO: similarity at the 4 amino acid level was 70%.
The invention provides an expression vector constructed by utilizing the tomato ascorbic acid biosynthesis gene PMI.
The invention provides a construction method of the expression vector, which comprises the following steps:
firstly, designing a full-length gene Primer by using Primer5, and amplifying in a PCR (polymerase chain reaction) instrument by using cDNA (complementary deoxyribonucleic acid) of tomato AC (alternating current) as a template to obtain a target fragment; linking the PCR product to a pEASY-B vector, transforming escherichia coli by a ligation product heat shock method, screening positive clones by a 50mg/L Km resistant plate, selecting the positive clones, carrying out shake culture on a shaker at 37 ℃ at 200r/min overnight, and selecting the positive clones after PCR detection by using gene specific primers for sequencing verification.
Secondly, selecting the clone shake bacteria with correct sequence comparison to extract plasmids; the recombinant plasmid is digested by Xbal and KpnI for 1.5 h.
Thirdly, cutting the gel, recovering the gel by using a gel recovery kit to obtain a target fragment, and connecting the target fragment to a pMV2 vector subjected to double enzyme digestion by Xbal and Kpn I by using T4 ligase; and (3) transforming escherichia coli by a ligation product heat shock method, selecting positive clones, carrying out PCR positive detection on the single clones by using a 35S binding gene reverse specific primer, selecting the positive clones, shaking the bacteria to extract plasmids, carrying out double enzyme digestion verification, and transferring the plasmids into agrobacterium-induced C58 cells by an electric shock method after the verification of the double enzyme digestion is correct.
Fourthly, selecting positive clones, carrying out shake culture at 150r/min on a shaking table at 28 ℃ overnight, and carrying out PCR positive detection on the bacterial liquid by using 35S plus gene reverse specific primers on the carrier; adding glycerol into the positive clone, mixing uniformly, and storing in a low-temperature refrigerator at-70 ℃.
In an embodiment of the invention, in the first step, the PCR amplification method comprises:
adopts a 10 mu L reaction system, uses EF1a as an internal reference and adopts Roche fluorescenceThe light quantitative PCR instrument LC480 performs measurement and analysis, and the system comprises
Figure RE-GDA0003729433390000131
Premix Ex Taq TM (2X) 5. mu.L, 0.5. mu.L each of forward and reverse primers, and 4. mu.L of template.
Each sample was replicated three times, the reaction procedure: pre-denaturation at 95 ℃ for 5 min; the specificity of PMI1 and PMI2 genes was determined by melting curve analysis after 45 cycles at 95 ℃ for 10s and 58 ℃ for 1 min.
The invention provides an application method for measuring relative expression quantity of leaves of a PPs (PPs) excess transgenic plant and ascorbic acid content, which comprises the following steps:
screening out the excessive transgenic plants with relatively high expression quantity of the T0 generation, and detecting and confirming the T1 generation again; 3 ultra-transgenic lines and non-transgenic lines of tender leaves are taken as materials, and the relative expression quantity is measured and analyzed by qPCR.
The content of ascorbic acid is measured by an enzyme-linked immunosorbent assay (ELISA) instrument by adopting a method of HCl extraction; the transformed plant young leaf is quick frozen in liquid nitrogen, the sample is ground into powder in the liquid nitrogen, and the powder is subpackaged with 0.1g of sample in a 2ml centrifuge tube, and each sample is repeated for 3 times.
During measurement, 1ml of pre-cooled 0.2mol/L HCl solution is added into each tube for extraction, about half an hour of extraction is carried out, the mixture is evenly mixed by inversion every 5min, and the mixture is centrifuged at 12000r/min at 4 ℃ for 10 min.
mu.L of the supernatant was added to 50. mu.L of 0.2mol/L NaH2PO4(pH 5.6) and the pH was adjusted to 5 and 6 with 0.2mol/L NaOH.
After mixing evenly, 100 mu L of supernatant is taken and added with 140 mu L of 0.12mol/L NaH in turn 2 PO 4 And 10. mu.L of 25mmol/L DTT, and reacted at room temperature for 30min in the dark. Taking 95 mu L of supernatant, adding 0.1ml of 0.2mol/L NaH 2 PO 4 And the value at the absorption wavelength of 265nm was measured by a microplate reader.
Finally, 5 mu L of 40U/ml AO enzyme is added, and the absorption wavelength value is measured after the reaction; the standard ascorbic acid solution is measured by a microplate reader, and a standard curve is drawn.
The invention is further described with reference to specific examples.
Examples
1) Plant material:
tomato material (Solanum lycopersicum) conventional line AC (stored in this laboratory) was used for gene cloning and genetic transformation. And (3) performing AC spring pot culture, collecting roots, stems, leaves and flowers of adult plants and fruit samples in different development periods after fruit setting and ripening, quickly freezing by using liquid nitrogen, and storing in an ultra-low temperature refrigerator at-70 ℃ for analyzing the tissue expression mode of PMIs.
2) PMIs tissue expression profile and assay of ascorbic acid content in each tissue:
qPCR primers for the PMI gene were designed using Primer5 software. A10- μ L reaction system was used, and EF1a was used as an internal control (Lovdal and Lillo,2009) for assay analysis by Roche fluorescence quantitative PCR LC480, which included
Figure RE-GDA0003729433390000151
Premix Ex Taq TM (2X) 5. mu.L, 0.5. mu.L each of forward and reverse primers, and 4. mu.L of template. Each sample was replicated three times, the reaction procedure: pre-denaturation at 95 ℃ for 5 min; melting curve analysis was performed after 45 cycles at 95 ℃ for 10s and 58 ℃ for 1min in order to determine the specificity of the primers. The relative gene expression was calculated using equation 2- Δ Ct (Svensson et al, 2006). And measuring the content of the ascorbic acid in each tissue of the tomato by adopting a universal high performance liquid chromatography.
3) PMIs gene amplification and sequence analysis:
tomato full-length cDNA sequences were obtained at SGN (http:// solgenomics. net/index. pl) using Unigene Solyc02g086090.2 and Solyc02g063220.2 as probes. And performing corresponding bioinformatics analysis on the PMI1 and the PMI2 by using software such as NCBI, ClustalW, Softberry, Multalin and the like. Gene specific primers containing the entire coding region were designed using Primer 5.0 software. And carrying out PCR amplification by using tomato strain AC leaf cDNA as a template.
4) Construction of PMI1 and PMI2 overexpression vector:
firstly, a Primer5 is utilized to design a full-length gene Primer, and a target fragment is obtained by taking cDNA of tomato AC as a template in a PCR instrument. Linking the PCR product to a pEASY-B vector, transforming escherichia coli by a ligation product heat shock method, screening positive clones by a 50mg/L Km resistant plate, selecting the positive clones, carrying out shake culture on a shaker at 37 ℃ at 200r/min overnight, and selecting the positive clones after PCR detection by using gene specific primers for sequencing verification. Selecting the clone with correct sequence alignment, shaking the bacterium and extracting the plasmid. The recombinant plasmid is digested by Xbal and KpnI for 1.5 h. The target fragment was recovered by a gel recovery kit, and the target fragment was ligated to the pMV2 vector digested with Xbal and Kpn I by using T4 ligase. And (3) transforming escherichia coli by a ligation product heat shock method, selecting positive clones, carrying out PCR positive detection on the single clones by using a 35S binding gene reverse specific primer, selecting the positive clones, shaking the bacteria to extract plasmids, carrying out double enzyme digestion verification, and transferring the plasmids into agrobacterium-induced C58 cells by an electric shock method after the verification of the double enzyme digestion is correct. Selecting positive clones, shake culturing overnight at 150r/min on a shaking table at 28 ℃, and carrying out PCR positive detection on the bacterial liquid by using 35S on the carrier and adding a gene reverse specific primer. And adding glycerol into the positive clone, mixing uniformly, storing in a low-temperature refrigerator at-70 ℃ and using for the genetic transformation of the tomato in the next step.
5) And (3) measuring the relative expression quantity of the PMIs transgenic plant leaves and the content of ascorbic acid:
and screening out the excessive transgenic plants with relatively high expression level in the T0 generation, and detecting and confirming the T1 generation again. 3 ultra-transgenic lines and non-transgenic lines of tender leaves are taken as materials, and the relative expression quantity is measured and analyzed by qPCR.
The ascorbic acid content was measured by a microplate reader using HCl extraction. The transformed plant young and tender leaves are quickly frozen by liquid nitrogen, the sample is ground into powder in the liquid nitrogen, and the powder is subpackaged with 0.1g of sample in a 2ml centrifuge tube, and each sample is repeated for 3 times. During measurement, 1ml of precooled 0.2mol/L HCl solution is added into each tube for extraction, about half an hour of extraction is carried out, the mixture is evenly mixed by inversion every 5min, and the mixture is centrifuged at 12000r/min at 4 ℃ for 10 min. mu.L of the supernatant was taken and 50. mu.L of 0.2mol/L NaH2PO4(pH 5.6) was added, the pH being adjusted between 5 and 6 with 0.2mol/L NaOH. After mixing, 100. mu.L of the supernatant was added with 140. mu.L of 0.12mol/L NaH2PO4(pH 7.5) and 10. mu.L of 25mmol/L DTT in this order, and reacted at room temperature in the dark for 30 min. 95. mu.L of the supernatant was added with 0.1ml of 0.2mol/L NaH2PO4(pH 5.6), the absorbance at 265nm was measured with a microplate reader, and finally 5. mu.L of 40U/ml AO enzyme was added, and the value was measured after the reaction. The standard ascorbic acid solution is measured by a microplate reader, and a standard curve is drawn.
In the present example, the PMI1 provided herein was aligned with PMI2 nucleic acid sequences as follows:
Figure RE-GDA0003729433390000161
Figure RE-GDA0003729433390000171
Figure RE-GDA0003729433390000181
in the embodiments of the present invention, the sequence alignment of PMI1 and PMI2 protein provided by the present invention is as follows:
Figure RE-GDA0003729433390000182
Figure RE-GDA0003729433390000191
in the present example, FIG. 2 is a graph showing the tissue expression profile of PMIs in different tissues of tomato and the total ascorbic acid content in different tissues of tomato, which is provided in the present example. Obtained by the expression profile of PMIs tissues and the determination and analysis of the content of ascorbic acid in each tissue in the step 2) of the example. And measuring the content of the ascorbic acid in each tissue of the tomato by adopting a universal high performance liquid chromatography.
FIG. 3 is a PCR assay of transgenic plants provided in the examples of the invention. Firstly, PMIs gene amplification and sequence analysis in the step 3) are carried out to obtain the full-length sequence of the PMIs gene; constructing PMI1 and PMI2 overexpression vectors through a step 4); detection of relative expression of leaves of transgenic plants with excess amount is obtained by detecting the relative expression of leaves of transgenic plants with excess amount of PMIs in step 5) of the example. The relative expression was analyzed by qPCR.
In the figure: m: labeling with molecular weight; n: negative control; p: positive control (recombinant plasmid); a:1 to 5 are PMI1 transgenic plants with excessive weight; b, 1 to 15 are PMI2 hypertransgenic plants; c, 1 to 14 are PMIs co-suppression transgenic plants; the primers used for PCR detection were 35S plus gene reverse primers.
FIG. 4 is a diagram showing the analysis of the expression of a target gene in leaves of a transgenic line according to the present invention. The expression of the PMIs in the step 5) of the embodiment is detected by the relative expression of the leaves of the transgenic plants with excess PMIs. The relative expression was analyzed by qPCR.
In the figure: o2, O11, O12, O17 and O18 are PMI1 excess strains; o4, O5, O6, O12, O14, O16, O17, O18, O22 and O26 are PMI2 excess strains; WT wild type.
FIG. 5 is the detection of the expression level of the fruit of transgenic line of overexpression PMI1 provided by the embodiment of the invention (A, C); measurement of AsA content in fruit of transgenic line overexpressing PMI2 (B, D). Obtained by measuring the ascorbic acid content of leaves of PMIs over-transgenic plants in the step 5) of the example. The ascorbic acid content was measured by a microplate reader using HCl extraction.
In the figure: WT wild type; o2-6, O11-7 and O12-2 are PMI1 over-plants; o4-2, O12-11 and O18-9 are PMI2 over-plants.
FIG. 6 is a graph showing the expression analysis of genes involved in AsA synthesis in leaves of PMIs over-transgenic lines according to the present invention. The expression of the PMIs in the step 5) of the embodiment is detected by the relative expression of the leaves of the transgenic plants with excess PMIs. The relative expression was analyzed by qPCR.
In the figure: WT wild type; o12-2: PMI1 superplants; o4-2: PMI2 hyperplantlets.
FIG. 7 is a map of the pMV2 vector provided by the present invention.
Proof section (examples/experiments/pharmacological analysis/positive experimental data, evidential material, identification reports, business data, development evidence, business collaboration evidence, etc. capable of demonstrating the inventive aspects of the present invention)
The implementation result shows that when PMI1 is overexpressed in tomato, the content of ascorbic acid in tomato fruits rises from 80mg/100gFW to 119mg/100gFW, and the rise reaches 48% (figure 5B). When PMI2 was overexpressed in tomato, the ascorbic acid content in tomato fruits rose from 80mg/100gFW to 112mg/100gFW, increasing by 40% (FIG. 5D). Thus, PMIs play an important promoting role in the accumulation of ascorbic acid in tomato fruits.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> Wuhanchu Biotech Co., Ltd
<120> tomato ascorbic acid biosynthesis gene PMI and application thereof
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Met Glu Met Glu Glu Gly Phe Lys Gly Leu Leu Arg Leu Ile Gly Ser
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Arg Leu Tyr Arg Leu Asn Ser Arg Thr Lys Ile Asp Glu Lys Gln Pro
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Tyr Ala Glu Phe Trp Met Gly Thr His Asp Ser Gly Pro Ser Tyr Ile
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Val Val Glu Arg Gly Gly Arg Ile Gln Asn Gly His Ala Asn Gly Gly
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Val Tyr Lys Asp Asp Asn His Lys Pro Glu Met Ala Leu Ala Leu Thr
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Ile Val Gln Thr Val Pro Glu Ile Val Glu Val Val Gly Asn Ala Leu
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Lys Leu Val Leu Arg Lys Leu Phe Thr Glu Ile Met Ser Ala Ser Lys
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Lys Asn Lys Val Arg Val Leu Thr Asp Lys Glu Gln Leu Val Leu Gly
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Leu Glu Lys Gln Tyr Pro Ser Asp Val Gly Val Leu Ala Ala Phe Leu
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Glu Ile Leu Gln Gly Thr Ala Val Asn Pro His Val Met Arg Tyr Ile
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Pro Pro Phe Asp Glu Phe Glu Val Asp Arg Cys Ile Leu Pro Glu Gln
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Ser Thr Thr Glu Phe Pro Ser Ile Pro Gly Pro Ser Ile Phe Met Val
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Val Glu Gly Glu Gly Thr Leu Thr Ser Ser Ser Asp Glu Ile Ile His
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Glu Gly Asp Val Leu Phe Ala Pro Ala Asn Thr Asn Ile Thr Val Ser
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435

Claims (6)

1. The tomato ascorbic acid biosynthesis gene PMI is PMI1 and PMI2, and the DNA of the ORF of PMI1 and PMI2 is SEQ ID NO: 1 and SEQ ID NO: 2; the nucleotide sequence of SEQ ID NO: 1 fragment is 1591 bp; the nucleotide sequence of SEQ ID NO: the 2 fragment is 1945 bp; the consensus at the PMI1 and PMI2 nucleic acid level was 79%.
2. A protein encoded by the tomato ascorbic acid biosynthesis gene PMI of claim 1, wherein the protein comprises the amino acid sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4; the nucleotide sequence of SEQ ID NO: 3 consisting of SEQ ID NO: 1 codes, 273 amino acids;
the nucleotide sequence of SEQ ID NO: 4 is represented by SEQ ID NO: 2 codes for 268 amino acids; the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: similarity at the 4 amino acid level was 80%.
3. An expression vector constructed using the tomato ascorbic acid biosynthesis gene PMI of claim 1.
4. A method for constructing the expression vector of claim 3, wherein the method comprises:
firstly, designing a full-length gene Primer by using Primer5, and amplifying in a PCR (polymerase chain reaction) instrument by using cDNA (complementary deoxyribonucleic acid) of tomato AC (alternating current) as a template to obtain a target fragment; linking a PCR product to a pEASY-B vector, converting escherichia coli by a ligation product heat shock method, screening positive clones by a 50mg/L Km resistant plate, selecting the positive clones, carrying out shake culture overnight at 200r/min on a shaker at 37 ℃, and selecting the positive clones after PCR detection by using a gene specific primer for sequencing verification;
secondly, selecting the clone shake bacteria with correct sequence comparison to extract plasmids; xbal and KpnI double enzyme digestion recombinant plasmid for 1.5 h;
thirdly, cutting the gel, recovering the gel by using a gel recovery kit to obtain a target fragment, and connecting the target fragment to a pMV2 vector subjected to double enzyme digestion by Xbal and Kpn I by using T4 ligase; connecting the products, transforming escherichia coli by a heat shock method, selecting positive clones, carrying out PCR positive detection on the single clones by using a 35S binding gene reverse specific primer, selecting the positive clones, shaking the bacteria to extract plasmids, carrying out double enzyme digestion verification, and transferring the plasmids into agrobacterium-induced C58 cells by an electric shock method after the verification is correct;
fourthly, selecting positive clones, carrying out shake culture at 150r/min on a shaking table at 28 ℃ overnight, and carrying out PCR positive detection on the bacterial liquid by using 35S plus gene reverse specific primers on the carrier; adding glycerol into the positive clone, mixing uniformly, and storing in a low-temperature refrigerator at-70 ℃.
5. The method for constructing an expression vector according to claim 4, wherein in the first step, the PCR amplification method comprises:
a10-microliter reaction system is adopted, EF1a is used as an internal reference, and the assay is carried out by a Roche fluorescence quantitative PCR instrument LC480, and the system comprises
Figure FDA0003601452070000021
Premix Ex Taq TM (2X) 5. mu.L of each forward and reverse primer, 4. mu.L of template;
each sample was repeated three times, the reaction procedure: pre-denaturation at 95 ℃ for 5 min; the specificity of PMI1 and PMI2 genes was determined by melting curve analysis after 45 cycles at 95 ℃ for 10s and 58 ℃ for 1 min.
6. The use of the tomato ascorbic acid biosynthesis gene PMIs of claim 1 in the measurement of the relative expression level and ascorbic acid content of leaves of PMIs transgenic plants with excess plant leaves, wherein the method for measuring the relative expression level and ascorbic acid content of leaves of PMIs transgenic plants with excess plant leaves comprises the following steps:
screening out the excessive transgenic plants with relatively high expression quantity of the T0 generation, and detecting and confirming the T1 generation again; taking 3 ultra-transgenic lines and non-transgenic lines as materials, and determining and analyzing relative expression quantity by qPCR;
the content of ascorbic acid is measured by an enzyme-linked immunosorbent assay (ELISA) instrument by adopting a method of HCl extraction; quickly freezing the transformed plant young and tender leaves with liquid nitrogen, grinding the sample in the liquid nitrogen into powder, subpackaging 0.1g of the sample in a 2ml centrifuge tube, and repeating each sample for 3 times;
during measurement, 1ml of precooled 0.2mol/L HCl solution is added into each tube for extraction, about half an hour of extraction is carried out, the mixture is inverted and uniformly mixed every 5min, and the mixture is centrifuged at 12000r/min at 4 ℃ for 10 min;
adding 50 μ L of 0.2mol/L NaH2PO4(pH 5.6) into 500 μ L of the supernatant, and adjusting the pH value to 5 and 6 with 0.2mol/L NaOH;
after mixing evenly, 100 mu L of supernatant is taken and added with 140 mu L of 0.12mol/L NaH in turn 2 PO 4 And 10. mu.L of 25mmol/L DTT, and reacted at room temperature for 30min in the dark. Taking 95 mu L of supernatant, adding 0.1ml of 0.2mol/L NaH 2 PO 4 Measuring the value of the absorption wavelength at 265nm by using an enzyme-labeling instrument;
finally, 5 mu L of 40U/ml AO enzyme is added, and the absorption wavelength value is measured after the reaction; the standard ascorbic acid solution is measured by a microplate reader, and a standard curve is drawn.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111560388A (en) * 2020-06-12 2020-08-21 武汉楚为生物科技股份有限公司 Gene for promoting synthesis of tomato ascorbic acid and application thereof
CN112301040A (en) * 2020-11-09 2021-02-02 华中农业大学 Gene for regulating and controlling accumulation of tomato ascorbic acid and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111560388A (en) * 2020-06-12 2020-08-21 武汉楚为生物科技股份有限公司 Gene for promoting synthesis of tomato ascorbic acid and application thereof
CN112301040A (en) * 2020-11-09 2021-02-02 华中农业大学 Gene for regulating and controlling accumulation of tomato ascorbic acid and application thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GENBANK: "XM_004232304.4", 《GENBANK》 *
GENBANK: "XM_004233441.4", 《GENBANK》 *
LI, X. J.等: "Biosynthetic Gene Pyramiding Leads to Ascorbate Accumulation with Enhanced Oxidative Stress Tolerance in Tomato", 《INT. J. MOL. SCI.》, vol. 20, pages 1 - 2 *
TAKANORI MARUTA等: "Arabidopsis Phosphomannose Isomerase 1, but Not Phosphomannose Isomerase 2, Is Essential for Ascorbic Acid Biosynthesis", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》, vol. 283, no. 43, pages 28842 *

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