A method for cultivating a plant with stay-green trait

Abstract

The present invention discloses a method for cultivating plants with stay-green traits, and relates to the BrCYP71 gene mutant and its application in stay-green of Chinese cabbage. The mutant of the present invention can inhibit the chlorophyll catabolism of plants, thereby maintaining stay-green, providing new gene resources for breeding, and also facilitating the further study of the molecular mechanism of stay-green.

Description

Technical Field

[0001] The present invention relates to the technical field of plant variety improvement, and particularly relates to a mutant gene / protein that can be used to improve plant phenotypes and its applications in improving plant phenotypes and cultivating improved varieties. Background Art

[0002] The sign of plant leaf senescence is often the change from green to yellow accompanied by a large amount of chlorophyll degradation and the coloring of other flavonoid substances. For leafy vegetables, during storage, transportation or processing, chlorophyll is easily degraded, and the leaves turn yellow and deteriorate, resulting in a decline in their edible quality and commercial quality. Therefore, delaying the senescence of leafy vegetable leaves, extending their shelf life, and increasing their economic value have become a difficult problem restricting production. Exploring more stay-green genes and better understanding the molecular mechanism of stay-green formation have great application prospects and economic value in the improvement of new varieties of leafy vegetables with better storage and transportation characteristics and the extension of shelf life.

[0003] According to physiological and photosynthetic characteristics, stay-green mutants are divided into functional and non-functional stay-green. Functional stay-green mutant leaves can remain green for a long time and maintain photosynthetic ability, which has important application value in agricultural production. For example, the maize variety FS854, sorghum variety R16, and rice variety SNU-SG1 all show high and stable yields. Non-functional stay-green mutant leaves have a basically unchanged or slowly decreasing total chlorophyll content during senescence, but the leaves lose the ability to photosynthesize. A large number of studies have shown that non-functional stay-green mutants are mainly regulated by chlorophyll catabolic enzymes or their encoding genes, such as NYC1, NOL, SGR, PPH, and PAO.

[0004] With the discovery of more and more stay-green mutants in different species, other causes of stay-green have gradually been revealed. Inhibiting the signal transduction of ethylene, abscisic acid, brassinolide, and strigolactone or activating cytokinin signal transduction can also lead to the stay-green trait. Some studies have shown that spraying exogenous hormones (ABA, ETH, and MeJA) on broccoli can accelerate yellowing and reduce chlorophyll content. This is due to the interaction between exogenous hormones and stay-green genes. In addition, some transcription factors, such as members of the NAC and WRKY families, have been reported to play a key role in inducing leaf senescence, and changes in the activity of these stay-green factors can also lead to stay-green. A non-functional stay-green mutant was found in pepper, and 11 hormone transduction-related transcription factors were discovered through transcriptome analysis. These transcription factors regulate chlorophyll degradation by regulating the expression of stay-green genes, resulting in the stay-green phenotype of pepper fruits.

[0005] In this study, a stay-green natural mutant nye2 was screened from the Chinese cabbage (Brassica rapa L. ssp. chinensis) cultivar Paopaqing. The phenotypic characteristics, genetic properties, and other physiological characteristics of nye2 were observed and measured. The stay-green gene BrCYP71 was identified by BSA-Seq and linkage analysis, and the function of BrCYP71 was verified by heterologous transformation in Arabidopsis thaliana. These results provide new gene resources for stay-green Chinese cabbage breeding. Summary of the Invention

[0006] The object of the present invention is to provide a Brcyp71 gene mutant and its application, especially the application in cultivating stay-green plants.

[0007] The above technical object of the present invention is achieved by the following technical solutions:

[0008] A Brcyp71 gene mutant, isolated and identified from the stay-green natural mutant nye2.

[0009] A Brcyp71 gene mutant, wherein the mutant is obtained by inserting GATC at the 253bp position in the first exon of BraA03g049920.3.5C, resulting in a frameshift mutation.

[0010] In some embodiments, the mutant is isolated and identified from the stay-green natural mutant nye2, specifically by inserting GATC at the 253bp position in the first exon of BraA03g049920.3.5C, resulting in a frameshift mutation.

[0011] In some embodiments, the gene mutant can encode a Brcyp71 protein mutant.

[0012] In some embodiments, the Brcyp71 protein sequence encoded by the gene mutant is as shown in SEQ ID NO: 4.

[0013] In some embodiments, the sequence of the gene mutant is as shown in SEQ ID NO: 3;

[0014] In some embodiments, the sequence of BraA03g049920.3.5C is as shown in SEQ ID NO: 1.

[0015] A Brcyp71 protein mutant, isolated and identified from the stay-green natural mutant nye2.

[0016] In some embodiments, the sequence of the protein mutant is as shown in SEQ ID NO: 4.

[0017] In some embodiments, the protein mutant is encoded by SEQ ID NO: 3.

[0018] Use of the Brcyp71 gene mutant and / or protein mutant in any of the above schemes for inhibiting plant leaf senescence.

[0019] In some embodiments, the inhibitory effect is achieved by down-regulating the expression level of chlorophyll catabolic genes.

[0020] In some embodiments, the inhibitory effect is achieved by inhibiting chlorophyll degradation.

[0021] In some embodiments, the inhibitory effect is achieved by up-regulating or maintaining the chlorophyll expression level.

[0022] In some embodiments, the chlorophyll catabolic genes include BrNYC1, BrNOL, BrHCAR, BrSGR1, BrSGR2, BrPPH, BrPAO, BrRCCR, and BrTIC55.

[0023] In some embodiments, the plant exhibits a stay-green phenotype.

[0024] Use of the Brcyp71 gene mutant and / or protein mutant in any of the above schemes for cultivating plants with improved chloroplast metabolism level.

[0025] In some embodiments, the method for cultivating plants includes expressing the Brcyp71 protein mutant as shown in SEQ ID NO: 4 in a recipient plant.

[0026] Provided is a method for cultivating plants with stay-green property, including any one of the following methods:

[0027] 1) Inactivating the activity of protein CYP71 in the plant;

[0028] 2) Reducing the activity of protein CYP71 in the plant, preferably, significantly reducing the activity;

[0029] 3) Inducing a frameshift mutation in the CYP71-encoding gene, which inactivates the encoded protein CYP71;

[0030] 4) Inducing a silent mutation in the CYP71-encoding gene;

[0031] 5) Knocking out the CYP71-encoding gene;

[0032] 6) Reducing the expression level of the CYP71-encoding gene, or,

[0033] 7) Use gene editing technology to insert GATC at the 253bp position in the first exon of BraA03g049920.3.5C of the recipient plant, causing a frameshift mutation;

[0034] 8) Transfer the Brcyp71 gene mutant into the recipient plant, and the chloroplast metabolism level of the plant is higher than that of the recipient plant;

[0035] 9) Any other conventional operations that can prevent the CYP71 coding gene from expressing or reduce the expression activity of the CYP71 protein.

[0036] In some embodiments, the CYP71 coding gene encodes the CYP71 protein as shown in SEQ ID NO: 4.

[0037] In some embodiments, the CYP71 coding gene is BraA03g049920.3.5C.

[0038] In some embodiments, BraA03g049920.3.5C is as shown in SEQ ID NO: 1.

[0039] In some embodiments, after the activity of the plant protein CYP71 is lost, the expression level of the chlorophyll catabolism gene is down-regulated, and the chlorophyll catabolism genes include BrNYC1, BrNOL, BrHCAR, BrSGR1, BrSGR2, BrPPH, BrPAO, BrRCCR and BrTIC55.

[0040] In some embodiments, the mutated sequence of the CYP71 protein is as shown in SEQ ID NO: 4.

[0041] In some embodiments, the stay-green is a stay-green trait.

[0042] In some embodiments, the stay-green refers to the characteristic that the chlorophyll in the senescent leaves of the plant does not degrade or degrades insignificantly, and remains green for a long time or even does not turn yellow at all.

[0043] In some embodiments, the plant is any plant that expresses the Brcyp71 gene.

[0044] In some embodiments, the plant is a Brassica plant.

[0045] In some embodiments, the plant is a leafy vegetable.

[0046] In some embodiments, the leafy vegetable is Chinese cabbage; preferably, it is wrinkled leaf black Chinese cabbage.

[0047] In some embodiments, the leafy vegetable is the Chinese cabbage variety Paopaoqing, also known as Suizhou Paopaoqing.

[0048] In some embodiments, the plant is Arabidopsis thaliana.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] First, this is the first time that BrCYP71 has been cloned and proposed to be related to stay-green, providing new gene resources for breeding and facilitating further research on the molecular mechanism of stay-green.

[0051] Second, the stay-green mutant gene can keep the senescent leaves from turning yellow, which can extend the shelf life, improve the commercial quality, and reduce storage and transportation losses. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 are the morphological characteristics of WT and nye2. The scale bar is 2.0 cm. 1A shows the comparison of the cotyledon colors of WT and nye2 at 12 days after sowing. 1B shows the comparison of the detached true leaves at 40 days after sowing. 1C shows the comparison of the bottom senescent true leaves at 55 days after sowing.

[0053] Figure 2 are the analysis results of photosynthetic pigment contents in WT and nye2 at different days after dark induction, including 3A) chlorophyll a, 3B) chlorophyll b, 3C) total chlorophyll, and 3D) the ratio of chlorophyll a to b. Asterisks indicate significant differences (*P < 0.05).

[0054] Figure 3 are the analysis results of the photosynthetic characteristics of WT and nye2 at different days after sowing. 3Aa: net photosynthetic rate (Pn), 3B: chlorophyll fluorescence parameter (maximum photochemical efficiency of PSⅡ in the dark, Fv / Fm).

[0055] Figure 4 are the transmission electron microscopy observations of the ultrastructure of chloroplasts in the leaves of WT and nye2. 4A 1-2 : ultrastructure of chloroplasts in WT at 35 days after sowing, 4B 1-2 : ultrastructure of chloroplasts in nye2 at 35 days after sowing, 4C 1-2 : ultrastructure of chloroplasts in WT at 90 days after sowing, d1-2) ultrastructure of chloroplasts in nye2 at 90 days after sowing. The magnification of 4A1-D1 is ×1.2k, and that of A2-D2 is ×12.0k. The scale bar in a1-d1 is 5.0 μm, and that in A2-D2 is 500 nm. SL: stromal lamella; P: osmiophilic granule; S: starch grain; L: lipid globule.

[0056] Figure 5It is the distribution of SNP-Index on the chromosome. 5A shows the phenotypic characteristics of 'FT' and nye2 at 55 days after sowing. 5B shows the phenotypic characteristics of the Y pool and S pool at 30 days after sowing. The scale bar is 3.0 cm. 5C-5D are the preliminary mapping based on BSA-Seq. 5C: The mapping result diagram of Grade Pool-Seq (GPS); 5D: The candidate region on chromosome A03. The abscissa is the position of SNP on the chromosome, and the ordinate is the corresponding Ratio value.

[0057] Figure 6 It is the genetic physical map and candidate gene analysis diagram of the stay-green gene. 6A is the fine mapping of the stay-green gene. The number of recombinant individuals between the marker and the stay-green gene is shown under the linkage map, and the genetic distance (cM) between adjacent markers is indicated between the numbers of recombinant individuals. 6B are the genes annotated in the Chinese cabbage database within the stay-green candidate region. The stay-green candidate genes were narrowed down to a 550 kb region, and 19 candidate genes were screened by combining BSA-Seq. The arrows indicate the length and direction of the genes.

[0058] Figure 7 It is the gene structure and sequence analysis diagram of BraA03g049920.3.5C. 7A is the gene structure of BraA03g049920.3.5C and the nye2 mutation site. 7B is the partial gene sequence alignment of BraA03g049920.3.5C in WT and nye2. 7C is the protein sequence alignment of BraA03g049920.3.5C in WT and nye2.

[0059] Figure 8 It is the transgenic function verification result of BrCYP71. 8A shows the morphology of 4-week-old Col-0, cyp71, pCYP71::BrCYP71 plants and pCYP71::Brcyp71 plants. The scale bar is 1.5 cm. 8B shows the total Chl content after 4 days of dark induction. Five replicates were taken, and the average value was used for multiple comparison analysis to calculate the significant differences.

[0060] Figure 9 It is the bioinformatics analysis of BrCYP71 protein. 9A shows the conserved domain of BrCYP71 and the position of amino acid mutations. 9B-9D are the three-dimensional conformation, secondary structure and hydrophilicity analysis of BrCYP71 in WT and nye2.

[0061] Figure 10 It is the gene expression analysis in WT and nye2. 10A shows the expression level of BrCYP71 in various organs of WT and nye2 at 90 days after sowing. 10B shows the expression level of CCGs in WT and nye2. Actin was used as an internal reference. The above data are the average values of 3 biological replicates, and the asterisks indicate significant differences (*P<0.05). Detailed implementation manners

[0062] The present invention will be described in detail below in conjunction with embodiments. However, the embodiments provided herein are for illustrative purposes only and are not intended to limit the present invention.

[0063] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods. The materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0064] Example 1: Identification of stay-green mutant phenotype

[0065] 1. Experimental materials and methods

[0066] 1.1 Plant materials

[0067] The stay-green mutant nye2 used in this study was a natural mutant discovered from the local variety Paopaoqing that had been self-crossed for 6 generations in 2019. The nye2 and its wild type (WT) were used to construct a population for genetic analysis. nye2 was crossed with the Chinese cabbage DH line 'FT' and then self-crossed to construct an F2 mapping population. All plants were grown in the solar greenhouse of Shenyang Agricultural University.

[0068] 1.2 Determination of chlorophyll content

[0069] At the seedling age of 30 days, 5 healthy WT and nye2 plants were selected each, and the third true leaf (from the outside to the inside) was selected for dark-induced senescence. The chlorophyll content of the leaves was measured at 0, 4, 8, 12, and 16 days of dark induction. The measurement was repeated 3 times and the average value was recorded.

[0070] 1.3 Determination of photosynthetic rate and chlorophyll fluorescence parameters

[0071] At the seedling age of 30 days, 5 healthy WT and nye2 plants were selected each, and the net photosynthetic rate (Pn) and chlorophyll fluorescence parameters of the fourth true leaf (from the outside to the inside) were measured using a Li-6800 portable photosynthesis analyzer. The measurement was carried out once every 3 days and repeated 3 times.

[0072] 1.4 Observation of chloroplast ultrastructure by transmission electron microscopy (TEM)

[0073] The senescent cotyledons 35 days after sowing and the senescent leaves 90 days after sowing were cut into strips (2-3 mm in length × 1 mm in width), immersed in 1 mL of glutaraldehyde solution, and evacuated to make them completely immersed. The samples were washed, fixed, dehydrated, embedded, stained, and the chloroplast ultrastructure was observed by TEM at magnifications of 1.2K and 12K respectively.

[0074] 2. Experimental results

[0075] At the seedling stage, the senescent cotyledons of nye2 were always green (Figure 1 A). At the adult stage, the bottom leaves of nye2 that naturally senesce always remain green until the leaves die. Figure 1 C). In contrast, the WT leaves turn yellow during the late growth stage as they senesce. After 48 h of dark induction, the detached leaves of nye2 remain green, indicating that the results of induced senescence and natural senescence are consistent. Figure 1 B). Throughout the growth process, nye2 exhibits a stable stay-green phenotype, while other phenotypes are not different from those of the WT. The mutant nye2 shows a stable stay-green phenotype.

[0076] The analysis results of the photosynthetic pigment contents of the WT and nye2 at different days after dark induction are shown in Figure 2 . During senescence, chlorophyll is gradually catabolized, and the contents of Chl a and Chl b gradually decrease, but the contents of Chl a and Chl b in nye2 are significantly higher than those of the WT. Figure 2 A, 2B). Figure 2 Figure C shows that after 16 d of dark induction, the total Chl content in the WT decreases, about 6% of that before dark induction, and the decrease rate is significantly faster than that of nye2. Figure 2 Figure D shows that the Chl a / b ratio in nye2 is higher than that of the WT, indicating that nye2 inhibits the degradation of Chl b. Therefore, the stay-green phenotype of nye2 may be caused by the delayed degradation of Chl b.

[0077] To analyze the type of stay-green of nye2, the Pn and Fv / Fm of the WT and nye2 were measured 30 d after sowing to determine whether nye2 can maintain photosynthetic capacity while staying green. Figure 3 ). The changing trends of the Pn and Fv / Fm of nye2 at different senescence stages are consistent with those of the WT, with no significant differences. The start time of the senescence process of nye2 is the same as that of the WT, and the senescence process of nye2 is not delayed. These results indicate that the photosynthesis time of nye2 is not extended, and the photosynthetic capacity is not improved. Therefore, nye2 is a non-functional stay-green mutant.

[0078] Chloroplasts are important organelles responsible for photosynthesis in plant cells, and the disintegration of chloroplasts is the earliest and most significant cellular structural change during the senescence process. Transmission electron microscopy was used to observe the ultrastructure of chloroplasts in senescent leaves to explore whether the impaired chlorophyll catabolism leads to abnormal chloroplast development. 35 d after sowing, the chloroplasts of the senescent cotyledons of nye2 are ellipsoidal, closely arranged along the cell wall, with large starch grains (S), and the stroma lamellae (SL) are clearly visible and closely arranged. Figure 4 B 1-2 ). However, in the stroma of the WT, the gap between the SLs increases, the stacking is uneven, the thylakoids begin to swell and partially disappear, and the degradation degree is higher than that of nye2. Figure 4A1 - A2). At 90 days after sowing, the chloroplasts in senescent leaves were significantly disintegrated, with significantly reduced volume, spherical in shape, lipid globules (L) appeared in the chloroplast stroma, and S and SL completely disappeared ( Figure 4 C - D). The envelope of WT chloroplasts fused, losing the complete structure, and some osmiophilic granules (P) and L were exocytosed from the chloroplasts ( Figure 4 C1). Compared with nye2, the number of P in WT was more, and the degree of senescence was also higher ( Figure 4 C2 - D2). These results indicate that the chloroplast structure of nye2 slowly disintegrates during senescence, resulting in non - functional stay - green.

[0079] Example 2: Identification of stay - green mutant genes

[0080] According to the phenotypic segregation ratios of each generation, the genetic characteristics of the stay - green trait of nye2 were identified (Table 1). The F1 plants obtained by reciprocal crosses all showed normal senescent yellowing phenotypes, indicating that this stay - green trait is controlled by a recessive gene. Among the backcross progeny of F1×nye2, 68 plants had the same phenotype as WT, 49 individuals were yellow, and 42 plants showed stay - green. The chi - square test results showed that the segregation ratio conforms to the Mendelian segregation ratio of 1:1. The yellow - green segregation ratio of the F2 population obtained by self - crossing F1 was 3.352:1, which was consistent with the expected value of the Mendelian segregation ratio of 3:1. Therefore, the stay - green candidate gene is controlled by a pair of recessive nuclear genes.

[0081] Table 1 Genetic analysis of the stay - green mutant nye2

[0082]

[0083] nye2 was crossed with 'FT' and then self - crossed to construct an F2 population ( Figure 5 A). From the F2 population, senescent yellowing and stay - green plants with obvious phenotypes were screened to construct two extreme pools, the Y pool and the S pool, for BSA sequencing ( Figure 5 B). 141,410,068 and 157,221,950 clean reads were obtained from the Y pool and the S pool respectively for further analysis. The base ratios of Q20 and Q30 in the two extreme pools were 97.88%, 97.58% and 94.23%, 93.61% respectively, for SNP index analysis ( Figure 5 C - D). The Radit test was used to analyze the differences between the two extreme pools. Low - quality SNPs / InDels were filtered, and sites with quality values greater than 100 were retained. SNP / InDel analysis located the stay - green gene within a 17.4 Mb interval on chromosome A03, and ED value analysis further narrowed the candidate interval to 10 Mb. To remove false positives and reduce background noise, a fixed window size of 400 kb was set, and the interval related to this trait was determined when the SNP / InDel ratio was high ( Figure 5C). Finally, the candidate interval was located on chromosome A03 ( Figure 5 D), within an interval of 2.0 Mb (Table 2), containing 344 genes.

[0084] Table 2 Statistics of trait-related regions

[0085] Chr Start End Length A03 24,000,015 25,194,639 1.2Mb A03 25,610,827 26,399,883 0.8Mb

[0086] Based on the preliminary mapping results of BSA-Seq, InDel markers were used to further fine-map the stay-green candidate genes. A total of 35 pairs of primers were designed, and 7 of them showed significant polymorphisms between WT and nye2 and could be used for linkage map construction. Using DNA denaturing polyacrylamide gel electrophoresis technology, 823 single plants with stay-green phenotypes in the F2 population were screened for recombinant single plants. The results showed that the candidate gene was located in a 550 kb region (A3: 25,729,824 - 26,279,566) between InDel 24 and InDel 29, containing 195 genes ( Figure 6 A). Using the Chinese cabbage reference genome, the sequence differences of all genes within the 550 kb interval between 'FT' and nye2 were compared by BSA-Seq, and 19 genes with mutation sites were identified ( Figure 6 B).

[0087] Functional annotation analysis was performed on the Arabidopsis homologous genes of the 19 candidate genes. The results showed that the functions of 16 genes were known or predicted, and the functions of 3 genes were unknown, but there was no research report on the association of these genes with the stay-green trait.

[0088] To exclude the gene sequence differences between Baopaqing and Chinese cabbage, whole-genome resequencing was performed on WT and nye2. The results showed that 18 mutation sites in the 550 kb interval were completely consistent with the BSA-Seq results. These 18 mutation sites were caused by interspecies differences. Therefore, BraA03g049920.3.5C was determined as the stay-green gene of nye2.

[0089] Primers were designed according to the genomic sequence to clone the full-length gene and CDS of BraA03g049920.3.5C in WT and nye2. The results showed that BraA03g049920.3.5C contained 2 exons and 1 intron, the full-length gene was 1547 bp, the CDS length was 579 bp, and it encoded 191 amino acids ( Figure 7 A, 7C).

[0090] > Nucleotide sequence of BraA03g049920.3.5C

[0091] ATGGTTCGGTCCTTTCACAAAGAGAGAGAAGAAGAGATTAATATTCTGATGGAAAAGTTGGAGCAAGCAAGTTCATCTTCTTCTACAGTAAACCTTAGCCAATTTTTTATCACCCTAACAAGCGATATAATGAGTAAAGTTGCCTTGGGAAGAAAATATAGTAGTGACGAAGGCACAGTCAATATCAAGACCGTAGTGAGGACGTTTTCTCGGGTATTTGGCACATTCCCTGTAGCGGAATACATTCCTAGTTTGGCATGGATAGATTGGATTCGCAGATTGGACGGTAAAGCAGAAGAAGTAAGTAAAACATTCGATGACTTTCTGGAAAAGGTGGTGCAAGAACATGATTTCGATGTAGATAAGAAAAGATCAGGTAGAAGACTATGTCCTGGAATCAGATTGGCATTGGTATTGGTTGAAGTGACAGTAGCAAACCTTGTGAAACGGTTTGACTGGAGAGTCCAGGTCGGACCTTATGGAGTTGATAAGCTTTATCTAGCCGAGGCAGCTGGTATAGAGGCATGTCGCAAATACCCTCTTATTGTCTTTCCAACTTCTGTTGTGTTCCCCATTTAA(SEQ ID NO:1)

[0092] >BraA03g049920.3.5C_amino acid sequence

[0093] MVRSFHKEREEEINILMEKLEQASSSSSTVNLSQFFITLTSDIMSKVALGRKYSSDEGTVNIKTVVRTFSRVFGTFPVAEYIPSLAWIDWIRRLDGKAEEVSKTFDDFLEKVVQEHDFDVDKKRSGRRLCPGIRLALVLVEVTVANLVKRFDWRVQVGPYGVDKLYLAEAAGIEACRKYPLIVFPTSVVFPI(SEQ ID NO:2)

[0094] Comparison of the sequences of WT and nye2 revealed that a 4-bp insertion (GATC) at position 253 bp in the first exon of BraA03g049920.3.5C in nye2 caused a frameshift mutation, resulting in the 98th amino acid being changed to the stop codon TAA, thereby leading to premature termination of translation( Figure 7 ), and loss of activity.

[0095] > nye2_nucleotide sequence

[0096] ATGGTTCGGTCCTTTCACAAAGAGAGAGAAGAAGAGATTAATATTCTGATGGAAAAGTTGGAGCAAGCAAGTTCATCTTCTTCTACAGTAAACCTTAGCCAATTTTTTATCACCCTAACAAGCGATATAATGAGTAAAGTTGCCTTGGGAAGAAAATATAGTAGTGACGAAGGCACAGTCAATATCAAGACCGTAGTGAGGACGTTTTCTCGGGTATTTGGCACATTCCCTGTAGCGGAATACATTCCTAGTGATCTTGGCATGGATAGATTGGATTCGCAGATTGGACGGTAAAGCAGAAGAAGTAAGTAAAACATTCGATGACTTTCTGGAAAAGGTGGTGCAAGAACATGATTTCGATGTAGATAAGAAAAGATCAGGTAGAAGACTATGTCCTGGAATCAGATTGGCATTGGTATTGGTTGAAGTGACAGTAGCAAACCTTGTGAAACGGTTTGACTGGAGAGTCCAGGTCGGACCTTATGGAGTTGATAAGCTTTATCTAGCCGAGGCAGCTGGTATAGAGGCATGTCGCAAATACCCTCTTATTGTCTTTCCAACTTCTGTTGTGTTCCCCATTTAA(SEQ ID NO: 3)

[0097] > nye2_nucleic acid sequence

[0098] MVRSFHKEREEEINILMEKLEQASSSSSTVNLSQFFITLTSDIM SKVALGRKYSSDEGTVNIKTVVRTFSRVFGTFPVAEYIPSDLGMD RLDSQIGR(SEQ ID NO: 4)

[0099] Example 3: Functional verification of stay-green mutant genes

[0100] BrCYP71 (BraA03g049920.3.5C) is highly homologous to Arabidopsis AT4G13290, and the Arabidopsis cyp71 mutant exhibits a stay-green phenotype ( Figure 8 A). During the senescence process, cyp71 leaves turned yellow later than Col-0, and the total Chl content was significantly higher than Col-0 at 4 days after dark induction ( Figure 8 ). By amplifying the CDS and promoter fragments of BrCYP71 in WT and nye2, pBWA(V)HS-BrCYP71 and pBWA(V)HS-Brcyp71 vectors were constructed and transferred into cyp71 plants using the Arabidopsis floral dip method. T3 transgenic positive plants were identified by resistance screening. During senescence, the leaf yellowing rate of pCYP71::BrCYP71 plants was the same as that of Col-0, and the total Chl content was not significantly different from that of Col-0 ( Figure 8 B), restoring the cyp71 plants to the wild-type phenotype ( Figure 8 A). However, the leaves of pCYP71::Brcyp71 plants still showed stay-green phenotype, and the total Chl content was not significantly different from that of cyp71 ( Figure 8 B). These results confirm the role of BrCYP71 in stay-green.

[0101] BrCYP71 was expressed in different tissues, including roots, stems, leaves, flowers, buds and pods, with the most significant difference in expression level in leaves. The expression level of BrCYP71 in nye2 leaves was 10% to 14% of that in WT ( Figure 10 A).

[0102] BrCYP71 protein is the 19th protein in cytochrome p450 (CYP) family 71 subfamily A. According to the conservative domain analysis of BraA03g049920.3.5C, the protein has a CYP conservative domain ( Figure 9 A). The CYP domain in BrCYP71 is located at amino acids 2-125, and the 98th amino acid mutation site in nye2 is located within the conserved domain ( Figure 9 A). BrCYP71 is a hydrophilic protein in both WT and nye2 ( Figure 9 D). However, compared with WT, the secondary structure of BrCYP71 in nye2 has a larger proportion of α-helices and a smaller proportion of extended strands and random coils ( Figure 9 D). The results of three-dimensional conformation analysis are consistent with those of secondary structure analysis ( Figure 9 B). Bioinformatics analysis showed that the frameshift mutation of BrCYP71 in nye2 affected the protein structure and caused its inactivity.

[0103] Since the change in chlorophyll content of nye2 during senescence was significantly different from that of WT, the expression levels of 9 chlorophyll catabolic genes (CCGs): BrNYC1, BrNOL, BrHCAR, BrSGR1, BrSGR2, BrPPH, BrPAO, BrRCCR, and BrTIC55 were analyzed by qRT-PCR. Total RNA from the roots, stems, leaves, flowers, buds, and pods of WT and nye2 was extracted using an RNA extraction kit as the reverse transcription template, and then reverse transcription was performed using a reverse transcription kit to obtain cDNA. The relative gene expression was calculated using the 2-ΔΔCt method, and significant analysis was performed using SPSS 26.0 software. Actin was used as an internal reference, and 3 replicates were set up.

[0104] The results showed that the expression levels of the 9 CCGs were significantly downregulated in nye2, while SGR, PAO, and NYC1 were hardly expressed ( Figure 10 B), indicating that chlorophyll degradation was inhibited during the senescence of nye2, resulting in a stay-green phenotype.

[0105] This specific embodiment is only an explanation of the present invention and is not a limitation thereof. Those skilled in the art can make modifications to this embodiment without creative contributions according to needs after reading this specification, but as long as it is within the scope of the claims of the present invention, it is protected by the patent law.

Claims

1. A method for cultivating a plant with stay-green trait, characterized in that, The activity of the protein CYP71 in the plant is lost, and the CYP71-encoding gene is BraA03g049920.3.5C; the stay-green property refers to the property that the chlorophyll in the senescent leaves of the plant does not degrade or degrades insignificantly, and remains green for a long time or even does not turn yellow at all. The method described includes any one of the following operations for cultivating the plant: (1) causing a frameshift mutation in the CYP71-encoding gene, which inactivates the encoded protein CYP71; the mutated sequence of the CYP71 protein is as shown in SEQ ID NO: 4; (2) causing a silent mutation in the CYP71-encoding gene; (3) knocking out the CYP71-encoding gene; or, (4) any other conventional operation that can prevent the CYP71-encoding gene from expressing the active protein CYP71. The BraA03g049920.3.5C is as shown in SEQ ID NO:

1. The plant is Chinese cabbage.

2. According to the method described in claim 1, after the activity of the plant protein CYP71 is lost, the expression levels of the chlorophyll catabolic genes are down-regulated, and the chlorophyll catabolic genes are BrNYC1, BrNOL, BrHCAR, BrSGR1, BrSGR2, BrPPH, BrPAO, BrRCCR, and BrTIC55.

3. According to the method described in claim 1, the plant is cultivated by transgenic technology.

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