Method for changing curly phenotype of arabidopsis thaliana leaf

Abstract

The invention relates to the field of gene engineering, in particular to a method for changing the curl phenotype of an arabidopsis thaliana leaf. According to the technical scheme, a Chinese cabbage BraATHB14-2 protein mutant coding gene is over-expressed in arabidopsis thaliana, and the mutant is obtained by mutating glycine into cysteine (SEQ ID NO: 3) or arginine (SEQ ID NO: 5) in the 197th amino acid of a protein with an amino acid sequence as shown in SEQ ID NO: 1.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a method for changing the curling phenotype of Arabidopsis thaliana leaves. Background Art

[0002] The Chinese cabbage gene BraATHB14-2 and the Arabidopsis gene ATHB14 (AT2G34710) belong to the HD-ZIPⅢ subfamily and are regulated by miR165 / 166 targets.

[0003] Itoh et al. reported that the phenotype of OSHB3 (homologous to ATHB14 and ATHB9) in rice was unchanged compared with the wild type; however, the complementary sequence of miR166 target was mutated at five sites on this gene, i.e., the bases complementary to the 2nd, 5th, 8th, 11th, and 17th bases counting from the 5' end of the 21-base mature miR166 sequence. Transgenic plants overexpressing the mutant gene OSHB3m showed curled or needle-shaped leaves. Rong et al. reported that two cucumber leaf curling mutants cul-1 and cul-2 were obtained by EMS mutagenesis. After MutMap positioning analysis, the CsPHB gene (Csa6G525430) exon5 (C mutated to T, Pro 194 Mutation to Leu 194 ), exon4 (G mutated to A, Gly 191 Mutation to Glu 191 ) caused by a non-synonymous mutation caused by a single base mutation on the CsPHB gene. The two mutation sites are located exactly at the cis-element position of the CsPHB gene targeted by miR166. Counting from the 5' end, the cul-1 and cul-2 mutation sites correspond to the 9th and 18th bases of the mature miR166 sequence, respectively. Jiang et al. reported that the EMS mutant M133 was located in the HB-D2 (Traes CS1D02G155200) gene of the HD-ZIPⅢ subfamily, and its single mutation site was located at the miRNA165 / 166 complementary site, corresponding to the 18th base of the mature miR166 sequence. The mutant M133 also showed the phenotype of wheat leaves curling upward.

[0004] Currently, the existing technology has not reported the mutation site of the wild-type BraATHB14-2 gene of Chinese cabbage that causes leaf curling. Summary of the invention

[0005] The object of the present invention is to provide a method for changing the curling phenotype of Arabidopsis leaves.

[0006] The method for changing the curling phenotype of Arabidopsis leaves according to the present invention comprises the following steps:

[0007] A gene encoding a mutant of the protein shown in SEQ ID NO: 1 is overexpressed in Arabidopsis thaliana, wherein the mutant of the protein shown in SEQ ID NO: 1 is obtained by mutating the 197th amino acid of the protein shown in SEQ ID NO: 1 from glycine to cysteine ​​(SEQ ID NO: 3) or arginine (SEQ ID NO: 5).

[0008] SEQ ID NO: 1: BraATHB14-2 protein sequence

[0009] MMVHTMHRESPDKGLDSGKYVRYTPEQVEALERVYTECPKPSSLRRQQLIRECPI

[0010] LSNIEPKQIKVWFQNRRCREKQRKEAARLQTVNRKLNAMNKLLMEENDRLQKQ

[0011] VSHLVYENGHMKHQIHTASGTTTTDNSCESVVVASGQQHQQQNPNPQHLQRDANN

[0012] PAGLL SIAKEALAEFLSKATGTAVDWVQMIGMKPGPDSIGIVAISRNCSGIAARACG

[0013] LVSLEPMKVAEILKDRPSWLRDCRSVDTLSVIPAGNGGTIELIYTQMYAPTTLAAA

[0014] RDFWTLRYTTCLEDGSYVVCEGSLSAATGGPTGPPSSNFVRAEMRPSGFLIRPCDG

[0015] GGSILHIVDHVDLDALSVPEVMRPLYESSKILAQKMTVAALRHVRQI AQETSGEVQ

[0016] YGGGRQPAVLRTFSQRLSRGFNDAVNGFVDDGWSPLGSDGAEDITLMINLSPGKF

[0017] GGNSFLPSFGSGVLCAKASMLLQNVPPAVLVRFLREHRSEWADYGVDAYAAASLR

[0018] ASPFAVPCARAGGLPSNQVILPLAQTVEHEEFLEVVRLEGHAYSPKDMGLARDMY

[0019] LLQLCSGVDEDVVGGCAQLVFAPIDESFADDAPLLPSGFRVIPLEPKSTSNNASVNR

[0020] TLDLASALEGSTRQGGEADPNGCHFRSVLTIAFQFTFDNHTRDNVASMARQYVRN

[0021] IVGSIQRVALAIAPRPDSSISPISAPTSPEALTLVRWISRSYRVHTGVDLFGSDSQTSG

[0022] DTLLHQVWSHTDAILCCSMKTNASPVFTFANQTGLDMLETTLVSLQDITLDKTLD

[0023] EPGRKALCSEFPKIMQQGYAHLPAGVCASSMGRLVSYEQATVWKVLEDDESNHCLAFMFVNWSFV*。

[0024] SEQ ID NO:2: CDS sequence of BraATHB14-2 gene

[0025] ATGATGGTCCACACGATGCACAGAGAGTCCCCGGACAAAGGGTTGGACTCCGG

[0026] CAAGTATGTTAGGTACACGCCTGAGCAAGTGGAAGCTCTTGAGAGAGTTTATAC

[0027] TGAGTGTCCTAAGCCCAGCTCTCTCAGAAGACAGCAACTCATACGAGAATGTC

[0028] CCATTCTCTCCAACATCGAGCCTAAACAGATCAAAGTTTGGTTTCAAAACCGCA

[0029] GATGTCGAGAGAAGCAGAGGAAAGAAGCTGCTCGTCTCCAGACAGTGAACAG

[0030] AAAGCTCAATGCAATGAATAAACTGCTGATGGAAGAGAATGATCGTCTGCAGA

[0031] AGCAAGTTTCTCACTTGGTCTATGAGAATGGCCACATGAAACACCAAATACACA

[0032] CTGCTTCAGGGACGACCACAGACAACAGCTGTGAGTCTGTGGTCGTAGCAAGT

[0033] GGTCAGCAACATCAACAGCAAAACCCAAATCCTCAGCATCTCCAACGAGATGC

[0034] TAACAACCCAGCTGGTCTCCTTTCAATAGCAAAGGAGGCTCTAGCAGAGTTCCT

[0035] TTCCAAGGCTACAGGAACTGCTGTTGACTGGGTTCAGATGATTGGGATGAAGC

[0036] CT G GTCCGGATTCTATTGGCATCGTAGCTATTTCCCGCAACTGCAGTGGAATTGC

[0037] CGCACGTGCCTGCGGCCTCGTGAGCTTAGAGCCCATGAAGGTTGCTGAAATCC

[0038] TCAAAGATCGTCCATCTTGGCTCCGTGATTGTCGAAGCGTGGACACTCTTAGCG

[0039] TGATTCCCGCTGGAAACGGTGGGACTATCGAGCTTATATACACTCAGATGTATGC

[0040] TCCAACAACGTTAGCAGCAGCTCGTGACTTCTGGACGCTGAGATACACCACTT

[0041] GTTTAGAAGATGGAAGCTATGTGGTTTGTGAGGGGTCGCTTAGTGCTGCAACTG

[0042] GTGGGCCTACTGGGCCGCCTTCTTCAAACTTTGTGAGAGCTGAGATGCGACCA

[0043] AGCGGGTTCCTCATCCGTCCTTGTGATGGTGGTGGTTCCATTCTCCACATTGTTG

[0044] ACCATGTTGATTTGGATGCGTTGAGTGTCCCTGAAGTCATGAGGCCTCTCTATG

[0045] AATCTTCAAAGATTCTTGCTCAGAAAATGACCGTTGCTGCGCTGAGACATGTAA

[0046] GACAGATTGCACAAGAGACAAGTGGAGAAGTACAGTACGGTGGAGGGCGGCA

[0047] ACCTGCTGTTTTAAGAACCTTTAGTCAAAGACTCTCTCGGGGGTTCAATGATGC

[0048] TGTTAATGGGTTTGTGGATGATGGATGGTCACCACTGGGTAGTGACGGTGCTGA

[0049] GGATATAACACTTATGATAAACTTGTCCCCTGGAAAGTTTGGTGGGAACTCATT

[0050] CCTCCCTAGCTTCGGTAGTGGTGTGCTTTGTGCCAAGGCATCTATGCTGTTACA

[0051] GAACGTTCCACCCGCTGTGCTGGTTCGTTTCCTTAGAGAACACCGCTCTGAGTG

[0052] GGCTGACTACGGCGTGGACGCTTATGCTGCTGCATCCCTCAGAGCGAGTCCTTT

[0053] CGCTGTTCCTTGCGCTAGAGCTGGTGGCTTACCTAGTAACCAAGTCATTCTCCC

[0054] TCTAGCTCAGACAGTTGAACATGAAGAGTTTCTTGAGGTGGTTAGACTCGAAG

[0055] GTCACGCTTACTCACCTAAAGACATGGGCTTAGCTAGGGATATGTACTTACTACA

[0056] GCTTTGTAGCGGTGTTGATGAAGATGTGGTTGGAGGTTGTGCACAGCTTGTCTT

[0057] TGCTCCTATAGACGAATCATTTGCTGATGATGCACCTTTGCTTCCTTCTGGCTTC

[0058] CGTGTCATACCTCTTGAACCAAAATCAACCTCGAACAATGCATCTGTGAACCGT

[0059] ACATTGGATCTAGCATCGGCGTTAGAAGGATCTACACGTCAAGGAGGTGAAGC

[0060] AGACCCAAACGGTTGTCACTTTAGGTCGGTGCTAACTATAGCGTTCCAGTTCAC

[0061] GTTTGATAACCACACAAGAGACAATGTTGCTTCGATGGCACGTCAGTACGTGA

[0062] GAAACATAGTTGGATCGATTCAGAGGGTTGCTCTAGCTATTGCTCCTCGTCCTG

[0063] ACTCTAGTATCAGTCCAATATCTGCTCCCACTTCCCCTGAAGCTCTCACTCTTGT

[0064] CCGTTGGATCTCTAGGAGTTACAGAGTTCACACTGGTGTAGATCTCTTTGGATC

[0065] TGATTCTCAAACCAGTGGTGACACATTGCTGCATCAAGTCTGGAGCCATACCGA

[0066] TGCAATCTTGTGCTGCTCCATGAAAACAAATGCTTCACCGGTTTTCACATTTGC

[0067] AAACCAAACCGGTTTAGACATGCTGGAAACTACTCTCGTGTCCCTTCAAGACAT

[0068] AACGTTAGACAAGACCTTAGACGAGCCTGGTCGTAAAGCTCTCTGCTCTGAGT

[0069] TCCCTAAGATCATGCAACAGGGCTATGCTCATCTTCCTGCGGGTGTATGTGCGTC

[0070] AAGCATGGGGAGATTGGTGTCTTACGAGCAGGCAACAGTGTGGAAAGTTCTTG

[0071] AAGACGACGAATCAAATCATTGCTTAGCGTTTATGTTCGTTAATTGGTCCTTCGTTTGA*.

[0072] SEQ ID NO:3: Amino acid G at position 197 of BraATHB14-2 protein sequence 197th Mutation to C 197th The mutant protein was named 215 protein.

[0073] MMVHTMHRESPDKGLDSGKYVRYTPEQVEALERVYTECPKPSSLRRQQLIRECPI

[0074] LSNIEPKQIKVWFQNRRCREKQRKEAARLQTVNRKLNAMNKLLMEENDRLQKQ

[0075] VSHLVYENGHMKHQIHTASGTTTTDNSCESVVVASGQQHQQQNPNPQHLQRDANN

[0076] PAGLL SIAKEALAEFLSKATGTAVDWVQMIGMKPCPDSIGIVAISRNCSGIAARACG

[0077] LVSLEPMKVAEILKDRPSWLRDCRSVDTLSVIPAGNGGTIELIYTQMYAPTTLAAA

[0078] RDFWTLRYTTCLEDGSYVVCEGSLSAATGGPTGPPSSNFVRAEMRPSGFLIRPCDG

[0079] GGSILHIVDHVDLDALSVPEVMRPLYESSKILAQKMTVAALRHVRQI AQETSGEVQ

[0080] YGGGRQPAVLRTFSQRLSRGFNDAVNGFVDDGWSPLGSDGAEDITLMINLSPGKF

[0081] GGNSFLPSFGSGVLCAKASMLLQNVPPAVLVRFLREHRSEWADYGVDAYAAASLR

[0082] ASPFAVPCARAGGLPSNQVILPLAQTVEHEEFLEVVRLEGHAYSPKDMGLARDMY

[0083] LLQLCSGVDEDVVGGCAQLVFAPIDESFADDAPLLPSGFRVIPLEPKSTSNNASVNR

[0084] TLDLASALEGSTRQGGEADPNGCHFRSVLTIAFQFTFDNHTRDNVASMARQYVRN

[0085] IVGSIQRVALAIAPRPDSSISPISAPTSPEALTLVRWISRSYRVHTGVDLFGSDSQTSG

[0086] DTLLHQVWSHTDAILCCSMKTNASPVFTFANQTGLDMLETTLVSLQDITLDKTLD

[0087] EPGRKALCSEFPKIMQQGYAHLPAGVCASSMGRLVSYEQATVWKVLEDDESNHCLAFMFVNWSFV*.

[0088] SEQ ID NO:4: Base 589 G of CDS sequence of BraATHB14-2 gene 589th Mutation to T 589th After sequencing, the mutant gene was named 215 gene.

[0089] ATGATGGTCCACACGATGCACAGAGAGTCCCCGGACAAAGGGTTGGACTCCGG

[0090] CAAGTATGTTAGGTACACGCCTGAGCAAGTGGAAGCTCTTGAGAGAGTTTATAC

[0091] TGAGTGTCCTAAGCCCAGCTTCTCCAGAAGACAGCAACTCATACGAGAATGTC

[0092] CCATTCTCTCCAACATCGAGCCTAAACAGATCAAAGTTTGGTTTCAAAACCGCA

[0093] GATGTCGAGAGAAGCAGAGGAAAGAAGCTGCTCGTCTCCAGACAGTGAACAG

[0094] AAAGCTCAATGCAATGAATAAACTGCTGATGGAAGAGAATGATCGTCTGCAGA

[0095] AGCAAGTTTCTCACTTGGTCTATGAGAATGGCCACATGAAACACCAAATACACA

[0096] CTGCTTCAGGGACGACCACAGACAACAGCTGTGAGTCTGTGGTCGTAGCAAGT

[0097] GGTCAGCAACATCAACAGCAAAACCCAAATCCTCAGCATCTCCAACGAGATGC

[0098] TAACAACCCAGCTGGTCTCCTTTCAATAGCAAAGGAGGCTCTAGCAGAGTTCCT

[0099] TTCCAAGGCTACAGGAACTGCTGTTGACTGGGTTCAGATGATTGGGATGAAGC

[0100] CT T GTCCGGATTCTATTGGCATCGTAGCTATTTCCCGCAACTGCAGTGGAATTGC

[0101] CGCACGTGCCTGCGGCCTCGTGAGCTTAGAGCCCATGAAGGTTGCTGAAATCC

[0102] TCAAAGATCGTCCATCTTGGCTCCGTGATTGTCGAAGCGTGGACACTCTTAGCG

[0103] TGATTCCCGCTGGAAACGGTGGGACTATCGAGCTTATATACACTCAGATGTATGC

[0104] TCCAACAACGTTAGCAGCAGCTCGTGACTTCTGGACGCTGAGATACACCACTT

[0105] GTTTAGAAGATGGAAGCTATGTGGTTTGTGAGGGGTCGCTTAGTGCTGCAACTG

[0106] GTGGGCCTACTGGGCCGCCTTCTTCAAACTTTGTGAGAGCTGAGATGCGACCA

[0107] AGCGGGTTCCTCATCCGTCCTTGTGATGGTGGTGGTTCCATTCTCCACATTGTTG

[0108] ACCATGTTGATTTGGATGCGTTGAGTGTCCCTGAAGTCATGAGGCCTCTCTATG

[0109] AATCTTCAAAGATTCTTGCTCAGAAAATGACCGTTGCTGCGCTGAGACATGTAA

[0110] GACAGATTGCACAAGAGACAAGTGGAGAAGTACAGTACGGTGGAGGGCGGCA

[0111] ACCTGCTGTTTTAAGAACCTTTAGTCAAAGACTCTCTCGGGGGTTCAATGATGC

[0112] TGTTAATGGGTTTGTGGATGATGGATGGTCACCACTGGGTAGTGACGGTGCTGA

[0113] GGATATAACACTTATGATAAACTTGTCCCCTGGAAAGTTTGGTGGGAACTCATT

[0114] CCTCCCTAGCTTCGGTAGTGGTGTGCTTTGTGCCAAGGCATCTATGCTGTTACA

[0115] GAACGTTCCACCCGCTGTGCTGGTTCGTTTCCTTAGAGAACACCGCTCTGAGTG

[0116] GGCTGACTACGGCGTGGACGCTTATGCTGCTGCATCCCTCAGAGCGAGTCCTTT

[0117] CGCTGTTCCTTGCGCTAGAGCTGGTGGCTTACCTAGTAACCAAGTCATTCTCCC

[0118] TCTAGCTCAGACAGTTGAACATGAAGAGTTTCTTGAGGTGGTTAGACTCGAAG

[0119] GTCACGCTTACTCACCTAAAGACATGGGCTTAGCTAGGGATATGTACTTACTACA

[0120] GCTTTGTAGCGGTGTTGATGAAGATGTGGTTGGAGGTTGTGCACAGCTTGTCTT

[0121] TGCTCCTATAGACGAATCATTTGCTGATGATGCACCTTTGCTTCCTTCTGGCTTC

[0122] CGTGTCATACCTCTTGAACCAAAATCAACCTCGAACAATGCATCTGTGAACCGT

[0123] ACATTGGATCTAGCATCGGCGTTAGAAGGATCTACACGTCAAGGAGGTGAAGC

[0124] AGACCCAAACGGTTGTCACTTTAGGTCGGTGCTAACTATAGCGTTCCAGTTCAC

[0125] GTTTGATAACCACACAAGAGACAATGTTGCTTCGATGGCACGTCAGTACGTGA

[0126] GAAACATAGTTGGATCGATTCAGAGGGTTGCTCTAGCTATTGCTCCTCGTCCTG

[0127] ACTCTAGTATCAGTCCAATATCTGCTCCCACTTCCCCTGAAGCTCTCACTCTTGT

[0128] CCGTTGGATCTCTAGGAGTTACAGAGTTCACACTGGTGTAGATCTCTTTGGATC

[0129] TGATTCTCAAACCAGTGGTGACACATTGCTGCATCAAGTCTGGAGCCATACCGA

[0130] TGCAATCTTGTGCTGCTCCATGAAAACAAATGCTTCACCGGTTTTCACATTTGC

[0131] AAACCAAACCGGTTTAGACATGCTGGAAACTACTCTCGTGTCCCTTCAAGACAT

[0132] AACGTTAGACAAGACCTTAGACGAGCCTGGTCGTAAAGCTCTCTGCTCTGAGT

[0133] TCCCTAAGATCATGCAACAGGGCTATGCTCATCTTCCTGCGGGTGTATGTGCGTC

[0134] AAGCATGGGGAGATTGGTGTCTTACGAGCAGGCAACAGTGTGGAAAGTTCTTG

[0135] AAGACGACGAATCAAATCATTGCTTAGCGTTTATGTTCGTTAATTGGTCCTTCGTTTGA*.

[0136] SEQ ID NO:5: BraATHB14-2 protein sequence amino acid G at position 197 197th Mutation to R 197th The mutant protein was named 216 protein.

[0137] MMVHTMHRESPDKGLDSGKYVRYTPEQVEALERVYTECPKPSSLRRQQLIRECPI

[0138] LSNIEPKQIKVWFQNRRCREKQRKEAARLQTVNRKLNAMNKLLMEENDRLQKQ

[0139] VSHLVYENGHMKHQIHTASGTTTTDNSCESVVVASGQQHQQQNPNPQHLQRDANN

[0140] PAGLLSIAKEALAEFLSKATGTAVDWVQMIGMKP R PDSIGIVAISRNCSGIAARACG

[0141] LVSLEPMKVAEILKDRPSWLRDCRSVDTLSVIPAGNGGTIELIYTQMYAPTTLAAA

[0142] RDFWTLRYTTCLEDGSYVVCEGSLSAATGGPTGPPSSNFVRAEMRPSGFLIRPCDG

[0143] GGSILHIVDHVDLDALSVPEVMRPLYESSKILAQKMTVAALRHVRQIAQETSGEVQ

[0144] YGGGRQPAVLRTFSQRLSRGFNDAVNGFVDDGWSPLGSDGAEDITLMINLSPGKF

[0145] GGNSFLPSFGSGVLCAKASMLLQNVPPAVLVRFLREHRSEWADYGVDAYAAASLR

[0146] ASPFAVPCARAGGLPSNQVILPLAQTVEHEEFLEVVRLEGHAYSPKDMGLARDMY

[0147] LLQLCSGVDEDVVGGCAQLVFAPIDESFADDAPLLPSGFRVIPLEPKSTSNNASVNR

[0148] TLDLASALEGSTRQGGEADPNGCHFRSVLTIAFQFTFDNHTRDNVASMARQYVRN

[0149] IVGSIQRVALAIAPRPDSSISPISAPTSPEALTLVRWISRSYRVHTGVDLFGSDSQTSG

[0150] DTLLHQVWSHTDAILCCSMKTNASPVFTFANQTGLDMLETTLVSLQDITLDKTLD

[0151] EPGRKALCSEFPKIMQQGYAHLPAGVCASSMGRLVSYEQATVWKVLEDDESNHCLAFMFVNWSFV*。

[0152] SEQ ID NO:6: Base G at position 589 of the CDS sequence of the BraATHB14-2 gene 589th mutated to C 589th and the resulting sequence, and the mutated gene is named the 216 gene.

[0153] ATGATGGTCCACACGATGCACAGAGAGTCCCCGGACAAAGGGTTGGACTCCGG

[0154] CAAGTATGTTAGGTACACGCCTGAGCAAGTGGAAGCTCTTGAGAGAGTTTATAC

[0155] TGAGTGTCCTAAGCCCAGCTCTCTCAGAAGACAGCAACTCATACGAGAATGTC

[0156] CCATTCTCTCCAACATCGAGCCTAAACAGATCAAAGTTTGGTTTCAAAACCGCA

[0157] GATGTCGAGAGAAGCAGAGGAAAGAAGCTGCTCGTCTCCAGACAGTGAACAG

[0158] AAAGCTCAATGCAATGAATAAACTGCTGATGGAAGAGAATGATCGTCTGCAGA

[0159] AGCAAGTTTCTCACTTGGTCTATGAGAATGGCCACATGAAACACCAAATACACA

[0160] CTGCTTCAGGGACGACCACAGACAACAGCTGTGAGTCTGTGGTCGTAGCAAGT

[0161] GGTCAGCAACATCAACAGCAAAACCCAAATCCTCAGCATCTCCAACGAGATGC

[0162] TAACAACCCAGCTGGTCTCCTTTCAATAGCAAAGGAGGCTCTAGCAGAGTTCCT

[0163] TTCCAAGGCTACAGGAACTGCTGTTGACTGGGTTCAGATGATTGGGATGAAGC

[0164] CT C GTCCGGATTCTATTGGCATCGTAGCTATTTCCCGCAACTGCAGTGGAATTGC

[0165] CGCACGTGCCTGCGGCCTCGTGAGCTTAGAGCCCATGAAGGTTGCTGAAATCC

[0166] TCAAAGATCGTCCATCTTGGCTCCGTGATTGTCGAAGCGTGGACACTCTTAGCG

[0167] TGATTCCCGCTGGAAACGGTGGGACTATCGAGCTTATATACACTCAGATGTATGC

[0168] TCCAACAACGTTAGCAGCAGCTCGTGACTTCTGGACGCTGAGATACACCACTT

[0169] GTTTAGAAGATGGAAGCTATGTGGTTTGTGAGGGGTCGCTTAGTGCTGCAACTG

[0170] GTGGGCCTACTGGGCCGCCTTCTTCAAACTTTGTGAGAGCTGAGATGCGACCA

[0171] AGCGGGTTCCTCATCCGTCCTTGTGATGGTGGTGGTTCCATTCTCCACATTGTTG

[0172] ACCATGTTGATTTGGATGCGTTGAGTGTCCCTGAAGTCATGAGGCCTCTCTATG

[0173] AATCTTCAAAGATTCTTGCTCAGAAAATGACCGTTGCTGCGCTGAGACATGTAA

[0174] GACAGATTGCACAAGAGACAAGTGGAGAAGTACAGTACGGTGGAGGGCGGCA

[0175] ACCTGCTGTTTTAAGAACCTTTAGTCAAAGACTCTCTCGGGGGTTCAATGATGC

[0176] TGTTAATGGGTTTGTGGATGATGGATGGTCACCACTGGGTAGTGACGGTGCTGA

[0177] GGATATAACACTTATGATAAACTTGTCCCCTGGAAAGTTTGGTGGGAACTCATT

[0178] CCTCCCTAGCTTCGGTAGTGGTGTGCTTTGTGCCAAGGCATCTATGCTGTTACA

[0179] GAACGTTCCACCCGCTGTGCTGGTTCGTTTCCTTAGAGAACACCGCTCTGAGTG

[0180] GGCTGACTACGGCGTGGACGCTTATGCTGCTGCATCCCTCAGAGCGAGTCCTTT

[0181] CGCTGTTCCTTGCGCTAGAGCTGGTGGCTTACCTAGTAACCAAGTCATTCTCCC

[0182] TCTAGCTCAGACAGTTGAACATGAAGAGTTTCTTGAGGTGGTTAGACTCGAAG

[0183] GTCACGCTTACTCACCTAAAGACATGGGCTTAGCTAGGGATATGTACTTACTACA

[0184] GCTTTGTAGCGGTGTTGATGAAGATGTGGTTGGAGGTTGTGCACAGCTTGTCTT

[0185] TGCTCCTATAGACGAATCATTTGCTGATGATGCACCTTTGCTTCCTTCTGGCTTC

[0186] CGTGTCATACCTCTTGAACCAAAATCAACCTCGAACAATGCATCTGTGAACCGT

[0187] ACATTGGATCTAGCATCGGCGTTAGAAGGATCTACACGTCAAGGAGGTGAAGC

[0188] AGACCCAAACGGTTGTCACTTTAGGTCGGTGCTAACTATAGCGTTCCAGTTCAC

[0189] GTTTGATAACCACACAAGAGACAATGTTGCTTCGATGGCACGTCAGTACGTGA

[0190] GAAACATAGTTGGATCGATTCAGAGGGTTGCTCTAGCTATTGCTCCTCGTCCTG

[0191] ACTCTAGTATCAGTCCAATATCTGCTCCCACTTCCCCTGAAGCTCTCACTCTGT

[0192] CCGTTGGATCTCTAGGAGTTACAGAGTTCACACTGGTGTAGATCTCTTTGGATC

[0193] TGATTCTCAAACCAGTGGTGACACATTGCTGCATCAAGTCTGGAGCCATACCGA

[0194] TGCAATCTTGTGCTGCTCCATGAAAACAAATGCTTCACCGGTTTTCACATTTGC

[0195] AAACCAAACCGGTTTAGACATGCTGGAAACTACTCTCGTGTCCCTTCAAGACAT

[0196] AACGTTAGACAAGACCTTAGACGAGCCTGGTCGTAAAGCTCTCTGCTCTGAGT

[0197] TCCCTAAGATCATGCAACAGGGCTATGCTCATCTTCCTGCGGGTGTATGTGCGTC

[0198] AAGCATGGGGAGATTGGTGTCTTACGAGCAGGCAACAGTGTGGAAAGTTCTTG

[0199] AAGACGACGAATCAAATCATTGCTTAGCGTTTATGTTCGTTAATTGGTCCTTCGTTTGA*.

[0200] Beneficial technical effects

[0201] The nucleotide sequence of the wild-type BraATHB14-2 gene of Chinese cabbage is shown in SEQ ID NO: 2. The present application study shows that the 589th base G of the CDS sequence of the wild-type BraATHB14-2 gene of Chinese cabbage589th Mutation to T 589th or C 589th , that is, the 197th amino acid of BraATHB14-2 is replaced by G 197th (Glycine Gly) mutated to C 197th (Cysteine ​​Cys) or R 197th (arginine Arg) can cause the leaves of overexpressing transgenic plants to curl upward stably. 589th When the gene remains unchanged, the phenotype of the overexpressing transgenic plants is that the leaves tend to be flat, which is consistent with the phenotype of wild-type Arabidopsis Col.

[0202] The curling of leaves inwards and their closure is one of the necessary physiological processes for the formation of cabbage heads, which directly determines the compactness and shelf life of cabbage heads and is a key factor affecting its commercial value. The mutation of this site causes the curling degree of leaves in the rosette stage to be 17 times that of wild-type leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0203] Figure 1 The sequencing map showing the successful mutation of the 589th base of the gene BraATHB14-2 and its point mutation gene;

[0204] Figure 2 Shown are the phenotypic analyses of overexpression of different BraATHB14-2 genes and mutants of Chinese cabbage in Arabidopsis. DETAILED DESCRIPTION

[0205] Example 1

[0206] 1.1 Mutation of base 589 of gene BraATHB14-2

[0207] The nucleotide sequence of the wild-type BraATHB14-2 gene of Chinese cabbage is shown in SEQ ID NO: 2. Using overlapping PCR technology and the CDS sequence (SEQ ID NO: 2) of the BraATHB14-2 gene as a template, the 215 gene and the 216 gene with a mutation at the 589th base were successfully cloned.

[0208] like Figure 1 As shown, compared with the gene BraATHB14-2, (1) point mutation gene 215: the 589th base of the gene BraATHB14-2 is changed from G 589th Mutation to T 589th , and the 197th amino acid of its protein sequence is replaced by G 197th (Glycine Gly) mutated to C 197th (Cysteine ​​Cys); (2) Point mutation gene 216: The 589th base of gene BraATHB14-2 changes from G to589th Mutation to C 589th , and the 197th amino acid of its protein sequence is replaced by G 197th (Glycine Gly) mutated to R 197th (ArginineArg).

[0209] Table 1 Primer list

[0210]

[0211] Example 2 Leaf phenotype changes of transgenic plants with overexpression of point mutation genes

[0212] After overexpression of these two point mutation genes in the Arabidopsis Col background, it was found that both CaMV35S::216 and CaMV35S::215 overexpressing plants showed a stable phenotype of leaf curling upward ( Figure 2 In the early stage of seedlings, some true leaves of 35S::BraATHB14-2 overexpressing plants curled slightly upward, but as they continued to grow, their leaves tended to flatten, which was consistent with the Col phenotype of wild-type Arabidopsis.

[0213] like Figure 2 As shown in Figures A, B, and D, qRT-PCR was used to detect the expression levels of BraATHB14-2 and its mutant genes in the overexpressed plants of CaMV35S::216, CaMV35S::215, and CaMV35S::BraATHB14-2, respectively. There were significant differences in their expression levels at the mRNA level and protein level. At the same time, Western blot analysis results showed that BraATHB14-2 or its mutant gene in these three transgenic plants was successfully expressed with GFP fusion expression protein. It can be seen that each transgenic plant was successfully created and its phenotype can be used for experimental comparative analysis.

[0214] These results indicate that the 589th base G of the Chinese cabbage gene BraATHB14-2 589th Mutation to T 589th or C 589th , that is, the 197th amino acid of BraATHB14-2 is replaced by G 197th (Glycine Gly) mutated to C 197th (Cysteine ​​Cys) or R 197th (arginine Arg), can cause the leaves of overexpressing transgenic plants to curl upward stably. 589th When the gene remains unchanged, the phenotype of the overexpressing transgenic plants is that the leaves tend to be flat, which is consistent with the phenotype of wild-type Arabidopsis Col.

Claims

1. A method for changing the curling phenotype of Arabidopsis leaves, characterized in that: The method comprises the following steps: The step of overexpressing a gene encoding a Chinese cabbage BraATHB14-2 protein mutant in Arabidopsis thaliana, wherein the amino acid sequence of the Chinese cabbage BraATHB14-2 protein mutant is shown in SEQ ID NO:3 or SEQ ID NO:

5.

2. The method for changing the curling phenotype of Arabidopsis leaves according to claim 1, characterized in that: The nucleotide sequence of the gene encoding the Chinese cabbage BraATHB14-2 protein mutant is shown in SEQ ID NO:4 or SEQ ID NO:6.

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