阅读说明：本技术 香蕉bZIP转录因子在调控果实发育过程中品质形成的应用及其表达载体构建 (Application of banana bZIP transcription factor in regulating and controlling quality formation in fruit development process and construction of expression vector thereof ) 是由 王笑一 李敬阳 魏卿 王安邦 李羽佳 许弈 于 2021-08-19 设计创作，主要内容包括：本发明提供香蕉bZIP转录因子在调控果实发育过程中品质形成的应用及其表达载体构建,该香蕉bZIP转录因子为香蕉MaHY5基因中的MaHY5like3基因；经对其表达特征和功能进行分析结果表明,MaHY5like3基因的表达随着香蕉果实的发育进程呈明显上调的趋势,MaHY5like3基因调控合成香蕉果实中淀粉和类胡萝卜素的MaAPS1和MaPsy2b基因的表达,从而调控果实品质的形成,为改良香蕉果实品质提供了候选基因,有利于为香蕉分子育种提供重要的理论基础。(The invention provides application of banana bZIP transcription factor in regulating and controlling quality formation in fruit development process and construction of an expression vector thereof, wherein the banana bZIP transcription factor is MaHY5like3 gene in a banana MaHY5 gene; the analysis result of the expression characteristics and functions of the gene shows that the expression of the MaHY5like3 gene is in a trend of being obviously up-regulated along with the development process of banana fruits, and the MaHY5like3 gene regulates and controls the expression of MaAPS1 and MaPsy2b genes of starch and carotenoid in synthesized banana fruits, so that the quality of the fruits is regulated and controlled, candidate genes are provided for improving the quality of the banana fruits, and an important theoretical basis is provided for molecular breeding of bananas.)

1. The application of the banana bZIP transcription factor in regulating and controlling the quality formation in the fruit development process is characterized in that: the banana bZIP transcription factor is MaHY5like3 gene in banana MaHY5 gene.

2. The use of the banana bZIP transcription factor of claim 1 to regulate quality development during fruit development, wherein: the nucleotide sequence of the MaHY5like3 gene is shown as SEQ ID NO: 1 is shown.

3. The use of the banana bZIP transcription factor of claim 1 to regulate quality development during fruit development, wherein: the application in regulating and controlling the content of starch and carotenoid in banana fruits.

4. The use of the banana bZIP transcription factor of claim 3 to regulate quality development during fruit development, wherein: for promoting the expression of the genes MaAPS1 and MaPsy2b of starch and carotenoid in the synthesized banana fruits.

5. The use of the banana bZIP transcription factor of claim 4 to regulate quality development during fruit development, wherein: the MaHY5like3 gene is bound to the G-box element of the MaAPS1 and MaPsy2b genes and is used for regulating the expression of the MaAPS1 and MaPsy2b genes.

6. A construction method of banana bZIP transcription factor expression vector is characterized in that: the method comprises the following steps:

(1) amplifying the CDS sequence of the MaHY5like3 gene by using a primer;

(2) and carrying out double enzyme digestion on the pCambia1300-35S-cGFP-Tnos vector plasmid by using two restriction endonucleases of XbaI and BamHI, recovering the enzyme digested vector and an amplified target fragment, connecting by using a NEBuilder HiFi DNA Assembly Master Mix kit, and carrying out transformation and sequencing verification to obtain the pCambia1300-35S-MaHY5like3-GFP expression vector of the banana transcription factor MaHY5like 3.

7. The method of claim 6, wherein the vector comprises: the primer sequence is as follows: 5'-AGAACACCTGCAGGTCGACTCTAGAATGCTCCAGGAACAAGCCACGAGCTC-3', and 5'-TGCTCACCATGGTACCCGGGGATCCCGCCGCCGCTTGGCTGTCTGCGGTGGCACTGCCAC-3'.

8. A construction method of a yeast expression vector of banana bZIP transcription factor is characterized in that: the method comprises the following steps:

(1) amplifying the CDS sequence of the MaHY5like3 gene by using a primer;

(2) carrying out double enzyme digestion on PB42AD vector plasmid by using EcoRI and XhoI restriction enzymes, recycling the enzyme-digested vector and the amplified target fragment, connecting by using a NEBuilder HiFi DNA Assembly Master Mix kit, and carrying out transformation and sequencing verification to finally obtain the PB42AD-MaHY5like3 yeast expression vector of banana transcription factor MaHY5like 3.

9. The method of claim 8, wherein the method comprises the steps of: the primer sequences were 5'-CATATGGCCATGGAGGCCAGTGAATTCATGCTGCAGGAGCAGGCAACCAGCTC-3' and 5'-AGTATCTACGATTCATCTGCAGCTCGAGCTACTTGCCTTCTCCATTGGCATTAA-3'.

Technical Field

The invention relates to the technical field of biology, in particular to application of a banana bZIP transcription factor in regulating and controlling quality formation in a fruit development process and construction of an expression vector thereof.

Background

The banana has rich nutrition and good taste, and is a fruit which is popular with consumers all over the world. The quality of banana fruits is an important factor influencing the quality and sales of bananas, and the improvement of the quality of the banana fruits is one of the main targets of banana production and breeding work. The quality of banana fruits is closely related to the accumulation of metabolites such as starch, sugar and carotenoid in vivo, so that the promotion of the accumulation of starch and carotenoid in the growth and development stages of banana fruits is very important for the formation of flavor and nutrient components of the ripe fruits. At present, the mechanism of regulation and control of banana fruit quality formation is unknown, the molecular mechanism research of synthesis and regulation of related metabolites in banana fruits is deeply carried out, the important theoretical basis is provided for banana molecular breeding, and no related report of banana bZIP transcription factor on quality formation in the process of regulating and controlling fruit development is found at present.

Disclosure of Invention

Therefore, the invention provides the application of the banana bZIP transcription factor in regulating and controlling the quality formation in the fruit development process and an expression vector construction method thereof.

The technical scheme of the invention is realized as follows:

the application of the banana bZIP transcription factor in regulating and controlling the quality formation in the fruit development process is disclosed, wherein the banana bZIP transcription factor is a MaHY5like3 gene in a banana MaHY5 gene.

Further indicates that the nucleotide sequence of the MaHY5like3 gene is shown as SEQ ID NO: 1 is shown.

Further illustrates the application of the banana bZIP transcription factor MaHY5like3 gene in regulating the content of starch and carotenoid in banana fruits.

Further, the banana bZIP transcription factor MaHY5like3 gene is used for promoting the expression of MaAPS1 and MaPsy2b genes for synthesizing starch and carotenoid in banana fruits.

Further, the banana bZIP transcription factor MaHY5like3 gene can be combined with the G-box element of MaAPS1 and MaPsy2b genes and used for regulating the expression of the MaAPS1 and MaPsy2b genes.

A construction method of banana bZIP transcription factor expression vector comprises the following steps:

(1) amplifying the CDS sequence of the MaHY5like3 gene by using a primer;

(2) and carrying out double enzyme digestion on the pCambia1300-35S-cGFP-Tnos vector plasmid by using two restriction endonucleases of XbaI and BamHI, recovering the enzyme digested vector and an amplified target fragment, connecting by using a NEBuilder HiFi DNAssembly Master Mix kit, and carrying out transformation and sequencing verification to obtain the pCambia1300-35S-MaHY5like3-GFP expression vector of the banana transcription factor MaHY5like 3.

Further, the primer sequence is as follows: 5'-AGAACACCTGCAGGTCGACTCTAGAATGCTCCAGGAACAAGCCACGAGCTC-3', and 5'-TGCTCACCATGGTACCCGGGGATCCCGCCGCCGCTTGGCTGTCTGCGGTGGCACTGCCAC-3'.

A construction method of a yeast expression vector of banana bZIP transcription factor comprises the following steps:

(1) amplifying the CDS sequence of the MaHY5like3 gene by using a primer;

(2) carrying out double enzyme digestion on PB42AD vector plasmid by using EcoRI and XhoI restriction enzymes, recycling the enzyme-digested vector and the amplified target fragment, connecting by using a NEBuilder HiFi DNAssembly Master Mix kit, and carrying out transformation and sequencing verification to finally obtain the PB42AD-MaHY5like3 yeast expression vector of the banana transcription factor MaHY5like 3.

Further illustrated, the primer sequences are 5'-CATATGGCCATGGAGGCCAGTGAATTCATGCTGCAGGAGCAGGCAACCAGCTC-3' and 5'-AGTATCTACGATTCATCTGCAGCTCGAGCTACTTGCCTTCTCCATTGGCATTAA-3'.

Compared with the prior art, the invention has the beneficial effects that: the invention obtains a bZIP transcription factor MaHY5like3 in banana MaHY5 gene which participates in quality formation in the growth and development process of banana fruits through screening, and analysis results of expression characteristics and functions of the gene show that the expression of the MaHY5like3 gene is in a trend of being obviously up-regulated along with the development process of the banana fruits, and the expression of genes related to synthesis of starch and carotenoid in fruits through transient over-expression of the transcription factor is up-regulated, so that the expression of the MaHY5like3 gene through regulating and controlling MaAPS1 and MaPsy2b genes for synthesizing starch and carotenoid in banana fruits is shown, and the quality formation of the fruits is regulated and controlled.

Drawings

FIG. 1 is a diagram of alignment of HY5 family transcription factor in banana A and B genomes and HY5 protein sequence and phylogenetic tree analysis in Arabidopsis, rice and maize genomes;

FIG. 2 is a diagram of analysis of the expression profile of the banana MaHY5 family gene of the present invention;

a is the expression pattern of the MaHY5 family gene in different tissues of bananas, and the statistical data are shown as follows: mean ± standard deviation, MaUBQ2 is the reference gene for qPCR;

b is the expression pattern of the MaHY5 family gene in different growth and development stages of banana fruits, and the statistical data are shown as follows: mean ± standard deviation, MaUBQ2 is the reference gene for qPCR;

FIG. 3 is a graph showing the comparison of the expression levels of MaHY5like3, MaAPS1 and MaPSY2b genes in fruits transiently overexpressed by the MaHY5like3 gene according to the present invention with those of a control group; the statistical data are expressed as: mean ± standard deviation, MaUBQ2 is the reference gene for qPCR;

FIG. 4 is a graph showing the results of the combination of MaHY5like3 protein and the G-box element of the promoters of MaAPS1 and MaPSY2b genes in the yeast single-hybrid test of the present invention.

Detailed Description

In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.

The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.

The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.

Example 1 identification and evolutionary analysis of the Banana MaHY5 family Gene

According to the invention, 5 genes MaHY5like and 4 genes MbHY5like are respectively identified from genome A and genome B of banana, and both genes have bZIP structural domains typical of HY5 family transcription factors, as shown in figure 1A. In order to further study the phylogenetic relationship of HY5 family members in bananas, the MEGA5 software was used to analyze the evolutionary relationship among HY5 proteins in bananas, Arabidopsis, rice and maize, all HY5 in bananas belongs to the same subfamily, and the relationship between Ma05_ p05820 in the A genome and Mba05_ g11630 in the B genome is closest to HY5 in Arabidopsis, as shown in FIG. 1B. The invention further researches 5 genes MaHY5 on the genome A, and the 5 genes Ma09_ p29200, Ma10_ p26990, Ma05_ p05820, Ma07_ p09800 and Ma06_ p06920 are named as MaHY5like1, MaHY5like2, MaHY5like3, MaHY5like4 and MaHY5like5 respectively.

Example 2 expression profiling of Banana MaHY5 family Gene

According to the invention, the RT-qPCR technology is adopted to detect the expression patterns of the 5 genes of the MaHY5 family in different tissues of banana plants and different stages of fruit development, and the results show that the gene of the MaHY5 family has different degrees of expression in each tissue, wherein the expression is highest in roots, and is second in bracts and fruits, as shown in FIG. 2A; along with the development process of banana fruits, the expression of the MaHY5like3 gene is in a clear up-regulation trend, the gene expression is down-regulated at the later fruit development stage, while the expressions of other four MaHY5 family genes do not show obvious regularity in the fruit development process, as shown in FIG. 2B, P1, P2, P3, P4 and P5 respectively represent fruits at 0 day, 10 days, 20 days, 40 days and 80 days after the buds of the bananas emerge; the suggestion that the MaHY5like3 protein may have important biological functions in fruit development.

The nucleotide sequence of the MaHY5like3 gene is as follows:

ATGCTCCAGGAACAAGCCACGAGCTCCCTTCCTTCCAGCAGCGAGAGATCCTCCAGCTCTGCCCCTCAGATGGAAATCAAAGAAGGAATGGAGAGCGACGAGGATGTAAGACGAGTGCCGGAGTTCGGGCTAGAGTTAGCAGGTCCGTCCTCCTCCGAGCGAGGACACGGTTCGGCGGTCGGCCAGGACCAGGCTCGGGTCGGGCAGCGGAGGAGGGGGAGGAGCCCCGCCGACAAAGAGCACAAGCGTCTCAAAAGGTTGCTGAGGAATAGAGTATCGGCTCAGCAGGCAAGGGAGCGGAAGAAAGCTTATCTGAATGATCTGGAGGCCAAGGTGAAGGATTTGGAGGCCAAGAACTCGGAGCTGGAGGAGAGGATGTCCACATTGCAGAACGAGAACAACATGCTGAGACAAATCCTGAAGAATACAACTGTGAGCAGAAGAGGATCCAGTGGCAGTGCCACCGCAGACAGCCAATAG。

example 3 functional analysis of MaHY5like3

In order to further research the function of MaHY5like3 in fruit development of bananas, an expression vector of 35S: MaHY5like3-GFP is constructed, and MaHY5like3 is transiently overexpressed in banana fruits subjected to bud picking for 20 days by an agrobacterium-mediated transient transformation method, and the expression level of the MaHY5like3 gene in the MaHY5like3 overexpressed fruits is analyzed by taking the fruits subjected to transient transformation of 35S-GFP as a control.

MaAPS1 and MaPsy2b are two genes respectively encoding key enzymes in the biosynthetic pathway of starch and carotenoid in bananas, and are two important genes determining the content of starch and carotenoid in banana fruits. The expression levels of both genes MaAPS1 and MaPsy2b were further analyzed by transient overexpression of MaHY5like3 in the fruits.

The construction of MaHY5like3 transient expression vector and the transformation of banana fruit slices are carried out by the following specific experimental methods:

a. the CDS sequence of the MaHY5like3 gene is amplified by using 5'-AGAACACCTGCAGGTCGACTCTAGAATGCTCCAGGAACAAGCCACGAGCTC-3' and 5'-TGCTCACCATGGTACCCGGGGATCCCGCCGCCGCTTGGCTGTCTGCGGTGGCACTGCCAC-3' primers;

b. carrying out double enzyme digestion on pCambia1300-35S-cGFP-Tnos vector plasmid by using two restriction endonucleases of XbaI and BamHI, recovering the enzyme digested vector and an amplified target fragment, connecting by using a NEBuilder HiFi DNA Assembly Master Mix kit, transforming and sequencing for verification, and finally obtaining the pCambia1300-35S-MaHY5like3-GFP expression vector of a banana transcription factor MaHY5like 3;

c. transferring the constructed vector into GV3101 agrobacterium tumefaciens, performing ice bath for 30min, performing liquid nitrogen for 5min at 37 ℃ for 5min, adding 1ml of LB liquid culture medium, and culturing at 28 ℃ and 220rpm for 4-5 h; sucking 200 μ L of bacterial liquid to LB solid culture medium of 20mg/L Kana and Rif, uniformly spreading on a flat plate, and performing inverted culture at 28 ℃ for 2 d; selecting a monoclonal for verification, transferring the correctly detected monoclonal agrobacterium into 5ml LB liquid culture medium, carrying out shaking culture at 28 ℃ and 220rpm for 24h, and keeping the bacteria for subsequent experiments;

d. transferring 1ml of the shaken bacterial solution into 500ml of liquid LB culture medium, and carrying out shaking culture at 28 ℃ and 220rpm for 16-24 h. The cells were collected by centrifugation at 4000rpm for 20 min. With a dip (1L dip containing 10mM Mgcl)₂10mM MES, 100. mu.M acetosyringone, pH 5.6), dispersing the cells, mixing them with a gun, and adjusting the OD of the bacterial solution₆₀₀When the value is 0.8-1, standing the staining solution at 24 ℃ for 3h, and immediately infecting the banana fruit slices;

e. cutting the Brazilian banana fruit slices to the thickness of 1mm on an ultraclean workbench, soaking for 1min by using 75% ethanol, and washing for 1 time by using sterile water; soaking in 20% sodium hypochlorite solution for 10min, and washing with sterile water for 3-5 times. Soaking the fruit slices in pCambia1300-35S-MaHY5like3-GFP and pCambia1300-35S-GFP infection solutions respectively, vacuumizing, and soaking for 15 min; after the thin sheet is taken out, bacteria liquid on the surface of the thin sheet is sucked dry by sterile filter paper, transferred to an MS culture medium, cultured in dark for 3 days at the temperature of 28 ℃, and cultured for 1 day under the light for expression analysis of genes of MaHY5like3, MaAPS1 and MaPsy2 b.

The results show that the expression level of the MaHY5like3 over-expressed fruit MaHY5like3 gene is significantly up-regulated compared with the control group (fruit transiently transformed with 35S-GFP), as shown in FIG. 3A. Moreover, the expression levels of both genes MaAPS1 and MaPsy2B were found to be significantly higher than the control group by transiently overexpressing MaHY5like3 in the fruits, as shown in FIGS. 3B and 3C, i.e., the expression levels of the genes MaHY5like3, MaAPS1 and MaPSY2B in the fruits transiently overexpressed by the gene MaHY5like3 were significantly higher than 35S: GFP transient material, further demonstrating that MaHY5like3 might regulate the formation of fruit quality by regulating the expression of related genes in the starch and carotenoid synthesis pathways.

Example 4 Yeast Monohybrid experiment

G-box, ACE-box, GATA-box and other elements are the binding sites of HY5 transcription factor, and HY5 binding elements are contained in the promoters of both MaAPS1 and MaPsy2b genes. According to the invention, the action relationship between the regulation and control of MaAPS1 and MaPsy2b by MaHY5like3 is verified through a yeast single hybrid experiment, and the specific steps are as follows:

(1) amplifying a CDS sequence of the MaHY5like3 gene by using 5'-CATATGGCCATGGAGGCCAGTGAATTCATGCTGCAGGAGCAGGCAACCAGCTC-3' and 5'-AGTATCTACGATTCATCTGCAGCTCGAGCTACTTGCCTTCTCCATTGGCATTAA-3' primers, carrying out double enzyme digestion on a PB42AD vector plasmid by using EcoRI and XhoI restriction endonucleases, recovering the enzyme digested vector and an amplified target fragment, connecting by using a NEBuilder HiFi DNA Assembly Master Mix kit, and carrying out transformation and sequencing verification to finally obtain a PB42AD-MaHY5like3 yeast expression vector of a banana transcription factor MaHY5like 3;

(2) carrying out double enzyme digestion on a target fragment and a pLacZ-2U vector by using KpnI and XhoI restriction endonucleases on the nucleotide sequences of the gene promoters of MaAPS1 and MaPsy2b, recovering the target fragment and the pLacZ-2U vector fragment after enzyme digestion, connecting by using T4 ligase, and carrying out transformation and sequencing verification to finally obtain vectors pLacZ-2U-MaAPS1-promoter and pLacZ-2U-MaPsy2b-promoter containing the gene promoters of MaAPS1 and MaPsy2 b;

(3) marking EGY48 frozen at-80 deg.C on SD-Ura plate, and culturing in 30 deg.C incubator for about 48 hr; jumping to obtain a single clone, putting the single clone into 5ml of YPDA liquid culture medium, placing the medium on a shaking table at 220rpm, and culturing the medium for about 16 hours at 30 ℃; transferring overnight cultured yeast into 50-250ml YPDA medium at a ratio of 1:200, and performing amplification culture for about 3 hr to OD₆₀₀0.5 plus or minus 0.1; subpackaging the yeast culture solution into 50ml BD tubes, and centrifuging at 1000g at room temperature for 5 minutes; discard the supernatant and use sterile ddH₂O resuspending the yeast cells; centrifuging at 1000g for 5 minutes at room temperature, and removing the supernatant; resuspending yeast cells with 1 XTE/LiAc;

(4) the vectors pLacZ-2U-MaAPS1-promoter and pLacZ-2U-MaPsy2b-promoter constructed with the promoters of MaAPS1 and MaPsy2b genes and the yeast expression vector PB42AD-MaHY5like3 constructed with the gene MaHY5like3 were mixed in equal amounts (usually 100ng for each vector) and 100ng of salmon sperm DNA was added; adding 100 μ L of 1 × TE/LiAc resuspended yeast cells, and mixing well; adding 600 mu L of PEG/LiAc solution, shaking and mixing uniformly; incubation at 30 ℃ and 200rpm for 30 minutes; adding 70 μ L of dimethyl sulfoxide (DMSO), and mixing by turning upside down; heat shock at 40 deg.C for 15 min; ice for 2 min; centrifuging at 14000g for 5s, and discarding the supernatant; 0.5mL of 1 XTE buffer was used to resuspend the transformed yeast cells;

(5) coating the resuspended yeast cells on an SD-Trp-Ura two-lacking plate, and culturing at 30 ℃ for 48-72 h; the grown single clones were streaked onto SD-Trp-Ura/Gal/Raf/X-Gal plates, incubated AT 30 ℃ for 12-24h, photographed, and used for detection of interaction SD-Leu-Trp-His +50mM 3-AT (3-Amino-1, 2, 4-triazole). A yeast single hybrid experiment shows that MaHY5like3 can be directly combined to G-box elements of MaAPS1 and MaPsy2b genes so as to participate in directly regulating and controlling the expression of the two genes, and as shown in figure 4, 1:1 is yeast stock solution; 1:100 is 100 times of yeast stock solution dilution; 1:1000 is 1000 times of yeast stock solution dilution.

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, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

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