CN116789779A

CN116789779A - Application of overexpression transcription factor StHB6 in improving potato traits

Info

Publication number: CN116789779A
Application number: CN202310599084.4A
Authority: CN
Inventors: 张凯; 吕长文; 杜叶; 都艺芝; 熊逸凡; 高艳; 杨朝彬; 韩永辉; 刘勋; 易小平; 唐道彬; 王季春
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2023-05-25
Filing date: 2023-05-25
Publication date: 2023-09-22

Abstract

The invention discloses application of an over-expression transcription factor StHB6 in improving potato traits, belongs to the technical field of plant molecular biology, wherein the amino acid sequence of the transcription factor StHB6 is shown as SEQ ID NO.22, the nucleotide sequence is shown as SEQ ID NO.21, and the over-expression transcription factor StHB6 in potatoes can improve the number of single plant tubers, the tuber forming rate, the total weight and the average weight of the potatoes and the resistance to HPV and late blight, and the research result provides important information for further revealing the functions of an HD-Zip family in potato growth and development, analyzing the molecular mechanism and regulating network of potato PVY and late blight resistance. Provides gene resources and new ideas for molecular breeding which further improves the yield, quality and disease resistance of potatoes.

Description

Application of overexpression transcription factor StHB6 in improving potato traits

Technical Field

The invention relates to the technical field of plant molecular biology, in particular to application of an overexpression transcription factor StHB6 in improving potato traits.

Background

The potato is the fourth largest grain crop after corn, rice and wheat, and has great significance for ensuring national grain safety. However, potato growth is susceptible to various diseases, and disease-resistant breeding has been an important breeding goal.

Transcription factors (Transcription factors, TF) are important participants in the regulation of gene expression networks. Related studies indicate that TF is a promising biotechnological target for improving plant disease resistance in genetic engineering, and in order to make full use of these immune-related TFs, a deeper understanding of their functions and mechanisms of action is required. While many studies have been conducted by predecessors on the growth and development of plants and abiotic stress by the HD-Zip I transcription factor, the mechanism of transcription factor HD-Zip I family members in the regulation of potato growth and development and disease resistance is not completely understood.

Disclosure of Invention

In view of the above, an object of the present invention is to provide an application of the overexpression transcription factor StHB6 in improving potato traits.

In order to achieve the above purpose, the present invention provides the following technical solutions:

1. use of the overexpressing transcription factor StHB6 for improving potato traits.

Preferably, the amino acid sequence of the transcription factor StHB6 is shown as SEQ ID NO. 22.

Preferably, the nucleotide sequence of the transcription factor StHB6 is shown as SEQ ID NO. 21.

The invention preferably discloses application of the overexpression transcription factor StHB6 in improving the single plant tuber number of potatoes.

The invention preferably discloses application of the overexpression transcription factor StHB6 in improving potato yield of potatoes.

The invention preferably relates to the use of the overexpression transcription factor StHB6 for increasing the total potato weight of potatoes.

The invention preferably provides for the use of the overexpressing transcription factor StHB6 for increasing the average potato weight of potatoes.

Preferably, the invention relates to the use of the overexpression transcription factor StHB6 for increasing HPV resistance of potato.

The invention preferably provides the use of the overexpressed transcription factor StHB6 for increasing the late blight resistance of potato.

In a preferred method of the invention, the method for over-expressing the transcription factor StHB6 is that the nucleotide sequence of the StHB6 in claim 3 is connected with pRC26B carrier skeleton through EcoR I and SpeI cleavage sites to obtain a recombinant expression vector, and then potato is transformed under the mediation of agrobacterium to obtain a transgenic plant over-expressing the transcription factor StHB6.

The invention has the beneficial effects that:

the research constructs the overexpression, interference and carrier of the StHB6, transforms potatoes, obtains the overexpression and interference transgenic plants, preliminarily identifies the regulation and control mechanism of the StHB6 in the growth and development of the potatoes and the resistance of PVY and late blight by measuring the physiological index and observing the phenotype of the StHB6 transgenic plants, and preliminarily analyzes the regulation and control network of the StHB6 and the molecular mechanism for regulating the growth and development and the disease resistance. StHB6 can inhibit the transcription of StTCTP by binding to the promoter of StTCTP, positively regulating plant resistance to PVY and late blight. The research result further reveals the functions of the HD-Zip family in potato growth and development, and analysis of potato PVY and late blight resistance molecular mechanisms and regulation networks provides important information. Provides gene resources and new ideas for molecular breeding which further improves the yield, quality and disease resistance of potatoes.

Drawings

In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:

FIG. 1 shows PCR amplification of StHB6 gene;

FIG. 2 shows analysis of sequences of StHB6 gene and encoded protein (A. Gene structure; B. Conserved domain; C. Potato StHB6 is aligned with Arabidopsis thaliana beta-branched homologous protein sequence, red horizontal line shows HD domain, yellow horizontal line shows LZ domain; D. Potato StHB6 is aligned with Arabidopsis thaliana HD-Zip I subfamily homologous sequence evolution tree);

FIG. 3 is a StHB6 promoter cis-acting element assay;

FIG. 4 shows the analysis of the transcriptional activity of StHB6 (A. Vector transformed AH109 yeast grown on SD/-Trp medium; B. Vector transformed AH109 yeast grown on SD/-Trp/-His/-Ade medium; C. StHB6 encoding protein has a full-length, N-terminal, C-terminal amino acid structure schematic (StHB 6 encoding region encodes 325 amino acids, stHB 6-N-terminal encoding protein 149 amino acids, stHB 6-C-terminal encoding protein 176 amino acids));

FIG. 5 shows analysis of StHB6 expression patterns (A. Predicted expression levels of StHB6 under each stress and hormone induction in potato database; B. Expression levels of StHB6 in 24h, 48h, 72h post-PVY inoculation leaves; C. Expression levels of StHB6 in 24h, 48h, 72h upper leaves post-PVY inoculation, data represent mean+ -SD, asterisks represent statistical significance, p <0.01, p <0.0001, analyzed with One-way ANOVA);

FIG. 6 shows the expression levels of StHB6 in potato tissues and organs (A. StHB6 expression levels in E3 tissues and organs, S1, S2, S4, S7 represent the respective periods of stolons; B. StHB6 expression levels in AC142 tissues and organs);

FIG. 7 is a pRC26B-HB6 vector map;

FIG. 8 shows StHB6 overexpression, interference vector construction and positive seedling acquisition (A. Schematic of the overexpression vector; B. Schematic of the interference vector; C. Resistant shoots and positive seedlings of potato pieces, stems; C. Schematic of the interference vector);

FIG. 9 shows the overexpression of StHB6 and the identification of interference transgenic plants (A. PCR identification of transgenic plants; B. RT-qPCR detection of the expression level of StHB6 in transgenic lines; C. Western Blot immunoblotting detection);

FIG. 10 shows agronomic trait statistics of StHB6 over-expression, interfering with plant subsurface 80d (A. Over-expression, interfering with plant subsurface phenotype; B. Over-expression, interfering with plant individual knot number; C. Over-expression, interfering with plant individual potato weight; D. Over-expression, interfering with plant average individual potato weight; E. Over-expression, interfering with plant potato type (aspect ratio), data represent mean.+ -. SD, asterisks represent statistical significance, <0.05, <0.0001, < p, < analyzed by One-way ANOVA,);

FIG. 11 shows agronomic traits of StHB6 over-expressed, interfering with the outdoor planting of the subsurface of the plant for 90d (A. Over-expressed, interfering with the subsurface phenotype of the plant; B. Over-expressed, interfering with the number of individual knots of the plant; C. Over-expressed, interfering with the individual weight of the plant; D. Over-expressed, interfering with the average individual weight of the plant; E. Over-expressed, interfering with the potato length of the plant; F. Over-expressed, interfering with the potato width of the plant; G. Over-expressed, interfering with the potato type (aspect ratio) of the plant, data represent mean.+ -. SD, asterisks represent statistical significance, p <0.05, p <0.0001 (analyzed by One-way ANOVA);

FIG. 12 is a graph of StHB6 overexpression, interference transgenic plant heterotrophic potato 60d (A. Overexpression, interference transgenic plant heterotrophic potato phenotype; B. Overexpression, interference transgenic plant heterotrophic potato length; C. Overexpression, interference transgenic plant heterotrophic potato width; D. Overexpression, interference transgenic plant heterotrophic potato aspect ratio value, data represent mean.+ -. SD, asterisks represent statistical significance, <0.05, <0.001 (analyzed by One-way ANOVA));

FIG. 13 is StHB6 subcellular localization following PVY infection;

FIG. 14 shows StHB6 overexpression, phenotype identification of interfering transgenic plants at PVY infection 21d (A. StHB6 overexpression, phenotype of interfering transgenic plants at PVY infection 21d inoculated leaves; B. StHB6 overexpression, phenotype of interfering transgenic plants at PVY infection 21d upper leaves; FIGS.);

FIG. 15 is DAB staining of wild-type and transgenic plant leaves at 3d of PVY infestation;

FIG. 16 is a graph showing the relative amounts of STHB6 and PVY-CP over-expressed at 4d and 11d for PVY infection, interference of the relative amounts of STHB6 in the leaves inoculated from the wild type plants for the plants, and B.PVY infection at 4d for the upper leaves of the transgenic plants, C.PVY infection at 11d for the upper leaves of the transgenic plants, D.PVY infection at 4d for PVY-CP in the leaves inoculated from the transgenic plants for STHB6, E.PVY infection at 4d for the upper leaves of the transgenic plants for PVY-CP for STHB6, F.PVY infection at 11d for the upper leaves of the transgenic plants for PVY-CP for STHB6, data representing mean.+ -. SD, asterisks representing statistical significance, p 0.05 x.p <0.0001 x < on-way > using One-A;

FIG. 17 is a graph showing the relative amounts of STHB6 and PVY-CP over-expressed at 21d for PVY infection, interfering with the relative amounts of STHB6 expressed in the leaves inoculated from each transgenic plant at 21d for PVY infection (A. PVY infection; B. PVY infection; C. PVY-CP expressed in leaves inoculated from each transgenic plant at 21 d; D. PVY infection; D. PVY-CP expressed in leaves upper from each transgenic plant at 21d for STHB 6; data represent mean.+ -. SD; asterisks represent statistical significance; p < 0.05; p <0.0001 (analyzed by One-way ANOVA)); the method comprises the steps of carrying out a first treatment on the surface of the

FIG. 18 shows post-leaf inoculation late blight 5d onset of StHB6 over-expressed and interfering strains (A. StHB6 over-expressed and interfering strains 'leaf inoculation late blight 5d phenotype; B. StHB6 over-expressed and interfering strains' leaf inoculation late blight 5d lesion diameter, data represent mean.+ -. SD, asterisks represent statistical significance, p <0.05, p <0.0001 (analyzed by One-way ANOVA);

FIG. 19 shows the results of trypan staining after 5d of a StHB6 overexpressing strain, an interfering strain leaf inoculated with late blight (A. StHB6 overexpressing strain, an interfering strain leaf inoculated with late blight 5d of trypan blue staining phenotype; B. StHB6 overexpressing strain, an interfering strain leaf inoculated with late blight 5d of diameter of trypan blue staining, data represent mean.+ -. SD, asterisks represent statistical significance, <0.05, <0.01 (analyzed by One-way ANOVA);

FIG. 20 is a StTCTP promoter with the binding motif of AtHB 6;

FIG. 21 is a graph of transient expression in Nicotiana benthamiana verifying binding of StHB6 to the StTCTP promoter;

FIG. 22 shows the relative expression levels of StHB6, PVY-CP, and StTCTP in inoculated leaves and upper leaves after PVY inoculation;

the relative expression of StHB6 in inoculated leaves after PVY 4d inoculation; relative expression of StHB6 in upper leaves after PVY 4d inoculation; STHB6 relative expression of upper leaves after PVY 11d inoculation; relative expression of StHB6 in upper leaves after PVY 21d inoculation; PCY-CP relative expression of inoculated leaves after 4d of PVY inoculation; relative expression of PCY-CP in upper leaves after 4d of PVY inoculation; relative expression of PCY-CP in upper leaves after 11d of PVY inoculation; relative expression of PCY-CP in upper leaves after 21d of PVY inoculation; sttctp relative expression of inoculated leaves after 4d of PVY inoculation; relative expression of sttctp in upper leaves after 4d inoculation with PVY; relative expression of sttctp upper leaves after 11d of PVY inoculation; relative expression of sttctp in upper leaves after 21d of PVY inoculation, data represent mean ± SD, asterisks represent statistical significance, p <0.05, p <0.0001 (analyzed with One-way ANOVA).

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the invention, so that those skilled in the art may better understand the invention and practice it.

Plant material used in this experimental study was wild-type detoxified Hubei potato No.3 (E3, tetraploid), diploid potato AC142, and Nicotiana benthamiana (Nicotiana benthamiana).

All potato materials and subsequently obtained transgenic lines of potato were cultivated in solid medium containing 4% sucrose MS, in 16h light, 8h darkness, at 23℃in a tissue culture room of potato. After the test tube plantlet grows for 2 weeks, the solid culture medium in the tissue culture plantlet is washed away, and the plantlet is acclimatized for 3 days in the same culture environment.

Primers used in the research are designed by utilizing SnapGene 3.2.1 software, and corresponding base sequences are added in front of required sequences according to different enzyme cutting sites of the constructed vector. The primer sequences and corresponding uses are shown in the table.

TABLE 1 primer sequences

EXAMPLE 1 cloning and bioinformatics analysis of Potato StHB6

StHB6 (Soltu. DM.05G021250) CDS sequences were obtained from the PGSC0003DMG400016790 sequence in the potato DM3-1 reference genome of the potato genome database Spud DB (V4.03), and the amplification primers StHB6-ORF-F/R (shown as SEQ ID NOS.1-2 in Table 1) were designed by Snap Gene. The StHB6 gene was cloned by PCR using the cDNA of E3 as a template. As shown in FIG. 1, a target fragment is obtained at about 1000bp, the target fragment is recovered by gel and then connected with a T vector, positive clones are detected by bacteria and sequenced, and whether the amplified sequence is consistent with the login sequence is detected. Sequencing results show that the CDS size of the amplified StHB6 gene is 978bp, and 325 amino acids are encoded. NCBI sequence numbering: LOC102592561, stHB6 gene CDS sequence is shown as SE Q ID NO.21, stHB6 gene coding amino acid sequence is shown as SE Q ID NO. 22.

Analysis of the chromosomal distribution of the StHB6 gene shows that this gene is located on chromosome 5 of potato. The gene structure was analyzed by GSDS software and the result is shown in FIG. 2, A, with 3 exons, 2 introns.

By analyzing the predicted result of the website Expasy, the protein coded by the StHB6 gene consists of 5026 atoms and has the molecular formula of C ₁₅₇₅ H ₂₄₄₉ N ₄₄₅ O ₅₄₇ S ₁₀ Isoelectric point PI is 4.66, molecular mass is 36691.02, total hydrophilic average (GRAVY) is-0.846, and the protein belongs to hydrophilic protein, instability coefficient is 48.07, and the protein belongs to unstable protein. The protein is shown to have no transmembrane domain, and has a Nuclear Localization Signal (NLS) at 109-117 (amino acids), conforming to the basic characteristics of StHB6 as a transcription factor.

The protein sequence of StHB6 gene was analyzed by comparison in NCBI database and Ensembl Plants database to obtain the gene sequence AtHB6 having the highest homology in Arabidopsis and potato. The protein sequence of the potato StHB6 gene was obtained from the potato database and the conserved domain of the StHB 6-encoding protein was predicted by NCBI and SMART (fig. 2, b). There is a DNA binding domain Homeodomain (HOX) at amino acids 62-115 that is involved in transcriptional regulation of key developmental processes, and HALZ (LZ) at amino acids 117-149, indicating that StHB6 belongs to the HD-Zip family (FIG. 2, C). Multi-sequence alignment of HD-Zip type I family sortilin, which has been reported in Arabidopsis, using MEGA, HB6 shows high similarity to members of the beta subclass of Arabidopsis with conserved homeobox domains and LZ structures, thus supposing that StHB6 belongs to the beta branch of the HD-Zip type I family in potato (FIG. 2, D).

Example 2 analysis of promoter sequence of potato StHB6 Gene

A2000 bp promoter sequence (NCBI sequence number: CP055238, SEQ ID NO. 23) upstream of a potato StHB6 gene is obtained from a potato database Spud DB, a promoter of StHB6 is amplified in a potato variety E3, the amplified promoter sequence is constructed on a PBI121-GUS vector, after sequencing and alignment, cis-acting element analysis is performed on the amplified promoter sequence by using an online analysis website PlantaCare, and as a result, as shown in FIG. 3 and Table 2, the sequence contains response elements such as low temperature, drought and biological stress and hormone response cis-acting elements such as Salicylic Acid (SA), abscisic acid (ABA), methyl jasmonate (MeJA), and the like, and the StHB6 possibly participates in biological processes such as biological stress and drought.

TABLE 2 StHB6 promoter cis-acting elements and related functional assays

Example 3 StHB6 transcriptional activation Activity assay

To verify whether StHB6 has transcriptional activity, we cloned the CDS full-length sequence of the gene, recombined onto pGBKT-7 vector, transferred to yeast strain AH109 according to yeast transformation method and empty pGBKT-7 respectively, plated on SD/-Trp medium for 3 days as shown in FIG. 4, A, and picked positive clone for culturing on SD/-Trp/-His/-Ade medium, and the full-length protein of the gene has transcriptional activity as shown in FIG. 4, B. To further confirm the functional region of the protein, the encoded protein was cut into two parts from the HD-Zip domain (62-149 amino acids), and the gene coding sequences of HB6-N (containing the HD-Zip domain) and HB6-C (not containing the HD-Zip domain) were constructed on pGBKT-7 vectors, respectively, and transformed into yeast strain AH 109. As a result, as shown in FIG. 4, C, the HB6-C region of StHB6 has transcriptional activity, and the HB6-N region containing the HD-Zip domain has no transcriptional activity. It was demonstrated that the StHB6 gene encodes a transcription factor having transcriptional activity and the C-terminal thereof has transcriptional activation activity.

Example 4 StHB6 Gene expression Pattern analysis

The transcriptome data analysis in the potato database Spud DB shows that the gene is subjected to Benzothiadiazole (BTH) and beta-aminobutyric acid (BABA) treatment to induce expression, and responds to SA signals, and the result is shown in FIG. 5A, and StHB6 is presumed to be possibly involved in response reaction of biological stress.

To determine the above results, we further analyzed the expression pattern of StHB6 by RT-qPCR at 24h, 48h, 72h of PVY treatment. As a result, as shown in FIG. 5, B, the expression level of StHB6 was changed in 72 hours after the PVY inoculation treatment, and the expression level of the inoculated leaf was highest at 24 hours, and as a result, as shown in FIG. 5, C, the expression level tended to decrease with the lapse of time, and the expression level of StHB6 was rapidly increased in the upper leaf of the inoculated leaf, and the expression level was continuously increased and maintained at a high expression level within 72 hours. The experimental results further indicate that StHB6 is expressed by induction of PVY.

As shown in FIG. 6A, the expression amount of StHB6 in the flower of tetraploid E3 was highest, and then the expression amount of the other tissues and organs was low, as shown in FIG. 6A, by analyzing the expression of StHB6 in each tissue and organ of potato by RT-qPCR. In addition, in order to obtain plants with higher editing efficiency, diploid potato AC142 is adopted for carrying out StHB6 gene editing, so that the expression of StHB6 in various tissues and organs of AC142 is also detected. As a result, as shown in FIG. 6, B, stHB6 was most expressed in tubers and leaves of AC142, unlike the expression pattern in tetraploid potato variety E3.

Example 5, over-expression, acquisition and identification of interfering transgenic lines

StHB6 expression pattern results suggest that potato StHB6 may be involved in the response to biotic stress, as well as in the development of various organs of potato. To investigate the specific function of StHB6 in potato in depth, we constructed StHB6 overexpression, interfering vectors, respectively. The full-length CDS of StHB6 was amplified by designing primers pRC26B-HB6-F/R (SEQ ID NOS.3-4) containing EcoR I and SpeI cleavage sites, and the detection by electrophoresis revealed a bright band around 1000 bp. The pRC26B vector backbone (pRC 26B-HB6 vector map shown in FIG. 7) was ligated by cleavage, E.coli DH 5. Alpha. Was transformed, colony PCR was performed, and the result was shown in FIG. 8, A, to obtain the overexpression vector pRC26B-3 xFLAG-StHB 6. Agrobacterium GV3101 was transformed for subsequent genetic transformation of potatoes. Specific 382bp in the StHB6 gene is selected as an interference fragment, and the primers shown in SEQ ID NO. 5-8 with Xho I and Xba I restriction sites are designed for amplification, wherein bright bands exist between 250 and 500 bp. The recombinant DNA is subjected to homologous recombination, escherichia coli DH5 alpha is transformed, colony PCR detection and sequencing are carried out, and the results are shown in FIG. 8 and B, so that an interference expression vector pHelsgate 8-StHB6 is obtained. Agrobacterium GV3101 was transformed for subsequent genetic transformation of potatoes.

And (3) utilizing an agrobacterium-mediated potato genetic transformation method to transform the StHB6 over-expression and interference vector into the potato. Through secondary root screening, as shown in fig. 8 and c, transgenic lines of 20 and 18 over-expressed and interference plants are obtained, the over-expressed plant is named as OE line, and the interference plant is named as Ri line. gDNA of the transgenic lines was detected using the overexpressing plant detection primer (35S-F/pRC 26B-HB 6-R) and the interfering plant detection primer (gat 8-HB 6-reverse-F/gat 8-HB 6-reverse-R), respectively. As shown in FIG. 9, A shows that the detection primers can be respectively amplified in gDNA of the transgenic strain to obtain specific bands of about 1000bp/250-500bp, the detection bands of the over-expressed plants OE-1, OE-3, OE-4 and OE-9 are clear, and the detection bands of the interference plants Ri-2, ri-4, ri-5, ri-8, ri-11 and Ri-12 are clear.

The RT-qPCR results are shown in figures 9 and B, and show that the expression quantity of the over-expressed transgenic lines OE-1, OE-3, OE-4 and OE-9 of StHB6 is obviously higher than that of the control, and the expression quantity of StHB6 in the interference transgenic lines Ri-2, ri-4, ri-5, ri-8, ri-11 and Ri-12 is obviously lower than that of the control. Wherein, the expression level of StHB6 in the over-expression strain is 11.31 times, 4.14 times, 5.12 times, and 2.85 times that of the control, respectively, and the expression level of StHB6 in the interference strain is 0.38 times, 0.197 times, 0.35 times, 0.124 times, 0.11 times, and 0.44 times that of the control, respectively. And selecting OE-1, OE-4 and OE-9 plants to perform Western Blot immunoblotting detection, and further determining the expression of the StHB6 protein level in the transgenic plants. As a result, as shown in FIG. 9 and C, the band of the target protein size was detected in each of the 3 transgenic plants.

Example 6 identification of the growth phenotype of transgenic lines

1. StHB6 negative control tuber weight

The agronomic traits of the tubers of the transgenic plants were examined, and the results are shown in FIG. 10, and the morphology and yield of the tubers of the transgenic plants after 80 days of greenhouse planting were observed. Compared with the wild type (E3), the results are shown in FIG. 10, A, the potato block morphology of all the transgenic lines (OE-1, OE-3, OE-4, ri-2, ri-4 and Ri-11) is not significantly different, and the number of single potato plants is not significantly different from that of the WT as shown in FIG. 10, B. As shown in FIGS. 10 and C, the individual potato weights of OE-3 and Ri-4 are remarkably reduced in 3 over-expression strains and 3 interference strains; as shown in figures 10 and D, the average individual potato weight of the over-expression strain OE-3 is remarkably reduced, while the average individual potato weight of the interference strain Ri-4 is not remarkably reduced; potato shapes as shown in fig. 10, e, there was no significant difference in potato shapes for all transgenic plants. Therefore, after 80 days of indoor planting of potatoes, the potato in the over-expression strain is changed again, the weight of the single potato is reduced, the average weight of the single potato is also reduced, and the result shows that StHB6 negatively regulates the weight of the tubers of the plants.

In order to verify the growth and development conditions of StHB6 transgenic plants under natural conditions, each transgenic plant is planted under natural illumination conditions in the experiment, and the tuber development conditions of the transgenic plants planted for 90 days are observed. The phenotype of the tubers of each strain is shown in FIG. 11, A, and there is no significant difference in tuber morphology for all transgenic strains (OE-1, OE-3, OE-4, OE-9, ri-2, ri-5, ri-8, ri-12). The number of single plant tubers is shown in figures 11 and B, and compared with a control, transgenic lines OE-3 and 9; the number of individual potatoes of Ri-2, 8 and 12 is obviously increased; as shown in FIGS. 11 and C, the transgenic strain OE-1 has significantly reduced individual potato weight and Ri-12 has significantly increased individual potato weight. As shown in FIGS. 11, D, the average individual potato weights OE-1, OE-9, and Ri-5 were significantly reduced. The potato shapes are shown in FIGS. 11, E-G, and there is no obvious difference in the potato shapes of the transgenes. These results show that under natural illumination conditions, the number of potatoes in the StHB6 over-expression strain and the number of potatoes in the interference strain are increased, but the weight of single potato in the over-expression strain is reduced, so that the average single potato weight is obviously reduced, and the result is consistent with the indoor potted planting result, thereby further indicating that the StHB6 negatively regulates the weight of potato tubers.

2. Heterotrophic potato growth of StHB6 transgenic plants

In order to further study the growth and development of potato tubers, a MS solid culture medium containing 8% of sucrose is used for inducing potato growth under the condition of short sunlight, tissue culture seedlings are inserted into the solid culture medium, 24 plants of each transgenic strain are respectively used for inducing potato growth for 90 days, and the potato length, the potato width, the aspect ratio, the potato growth rate and the average potato weight of test-tube potatoes are observed. As shown in fig. 12, a, the number of potatoes was increased in the interference strain compared to the control strain. As shown in 12, b, the potato length of the interfering lines was significantly increased compared to the control. As shown in fig. 12, c, the potato width of the interfering lines was significantly increased compared to the control. The potato shape is shown in FIGS. 12 and D, and the potato shape (aspect ratio) of each transgene is unchanged. As shown in Table 3, the heterotrophic potato yield, average potato weight of the interfering strain are higher than those of the control and over-expressed plants, and the growth and development of the interfering strain are higher than those of the control, so that StHB6 can negatively regulate the growth of potato tubers.

TABLE 3 statistics of StHB6 overexpression, interference of heterotrophic tubers from lines

EXAMPLE 7 PVY resistance analysis of Potato StHB6 transgenic plants

1. StHB6 subcellular localization changes before and after PVY infection

As a result, stHB6 was localized to the nucleus as shown in FIG. 12. 7 days after PVY virus inoculation of Nicotiana benthamiana, stHB6-GFP was transiently expressed in leaves of Nicotiana benthamiana by Agrobacterium-mediated methods. The results are shown in FIG. 13, in which green fluorescence of GFP-EV (empty) fills the whole cell membrane and nucleus; while the StHB6-GFP fluorescence signal fills the entire cell, indicating that upon PVY infection, the subcellular localization of StHB6 is altered, possibly localized to the nucleus and cytoplasm.

2. Phenotypic observation of StHB6 transgenic lines after PVY infection

To further investigate the function of StHB6 in PVY infection response, the present study used PVY to inoculate wild-type controls (WTs) and overexpression, interfering transgenic lines of StHB6, respectively. The results showed that after 21 days of PVY infection, the upper leaves of the StHB6 overexpressing lines showed some resistance to PVY infection, less leaf damage and fewer lesions compared to the inoculated leaves of the plants (FIG. 14, A) and to WT (FIG. 14, B); the interference plants show obvious susceptibility, the leaves have more obvious necrotic spots, and the damage degree is obviously worse than that of the control plants. We speculate that overexpression of StHB6 in potato may increase plant resistance to PVY.

3. DAB staining of potato StHB6 transgenic line leaves after PVY infection

After the potato is infected by PVY, a great amount of Reactive Oxygen Species (ROS) molecules are generated at the infection site in a short time, so that the cell death is caused, the growth of pathogens is limited to a certain range, the diffusion of the pathogens can be prevented, and the generation of a large range of injury is prevented. After 3 days of PVY inoculation, as shown in FIG. 15, after DAB staining of the inoculated and upper leaves of StHB6 overexpressing, interfering transgenic plants and wild-type control plants, a small range of light red stained areas was observed in WT and interfering leaves, indicating that PVY resulted in low levels of H after inoculation ₂ O ₂ Accumulation, failure to produce hypersensitivity reactions, the pathogen can continue to infect surrounding cells and eventually wilt the leaves. In contrast, some dark red colored portions were observed on leaves of the StHB6 overexpressing strain, indicating that infection with PVY may cause H ₂ O ₂ The large accumulation of these infected cells causes programmed death in a short period of time, inhibiting viral replication and preventing PVY from continuing to infect surrounding cells. The above results indicate that StHB6 may upregulate potato resistance to PVY.

4. Quantitative detection of StHB6 transgenic strain virus accumulation after PVY infection

To further determine the change in virus accumulation in StHB6 transgenic plants after PVY infection, we used RT-qPCR to detect the expression of PVY Coat Protein (CP) genes in inoculated and upper leaves infected for 4 days and upper leaves infected for 11 days, respectively. As shown in FIGS. 16, A-C, stHB6 was differentially expressed in overexpressing and interfering plants at day 4 and day 11 of PVY inoculation of transgenic plants and wild type controls. As shown in FIGS. 16 and D, it can be seen that the PVY CP genes in the leaves of the over-expressed and interference-expressed plants have a certain expression level, which indicates that PVY accumulates in the inoculated leaves. From FIG. 16, E, PVY had accumulated in the upper leaves 4 days after inoculation, but the amount of virus accumulated in the upper leaves of the StHB6 overexpressing plants was significantly reduced compared to WT, and the amount of virus accumulated in the upper leaves of the StHB6 interfering plants was significantly increased compared to WT. As shown in FIG. 16, F, the accumulation of PVY in the plants overexpressing StHB6 was extremely reduced in the upper leaves at 11 days of inoculation, indicating that an increase in the expression level of StHB6 delayed the spread and accumulation rate of the virus, while a decrease in the expression level of StHB6 contributed to the spread and accumulation of PVY.

As shown in FIGS. 17A and B, when the plants in which StHB6 is overexpressed and disturbed in the PVY 21 days were inoculated, the relative expression amounts of StHB6 in the inoculated leaves and upper leaves were examined. As shown in FIG. 17, C, after 21 days of virus inoculation, it was found that there was a certain amount of PVY CP gene expression in leaves of both over-expressed and interference-expressed plants, indicating that PVY accumulated in inoculated leaves. As shown in fig. 17, d, the accumulation of virus in the upper leaves of the interfering plants was significantly increased compared to WT at day 21 of virus inoculation, whereas the accumulation of virus in the upper leaves in the overexpressed plants was significantly decreased compared to WT. In summary, it can be shown that StHB6 participates in the transmission pathway of PVY, the accumulation amount of virus is significantly increased after the expression amount of StHB6 is reduced, and the accumulation amount of virus is significantly reduced after the expression amount of StHB6 is increased, and the increase of the expression amount of StHB6 can delay the transmission and accumulation speed of PVY virus and positively regulate the infection of PVY.

Example 8 control of StHB6 resistance to potato late blight

1. Patch assay

According to the stage of the onset of late blight, as shown in FIG. 18, A, after inoculation of late blight pathogenic bacteria, leaves of StHB6 over-expression strain can see a small amount of black lesions, and the lesions show needle eye size of not more than 5mm, belonging to stage 1-2 of stage of late blight onset. StHB6 interference plant leaf presents large black dead disease spots, the size of the disease spots is 8-15mm, white mycelium appears on the black dead disease spots, and the mycelium belongs to the grade 3-4 of the stage of late blight disease condition. As shown in FIG. 18, B, the lesion diameter of leaf blades of the StHB6 overexpressing strain is remarkably reduced compared with WT, while the lesion diameter of the StHB6 interfering strain is remarkably increased compared with WT, which indicates that the increase of the expression level of the StHB6 gene can inhibit the propagation of late blight spores, and the decrease of the expression level of StHB6 accelerates the propagation speed of late blight pathogenic spores. Taken together, the results indicate that StHB6 may be involved in the positive regulation of late blight resistance.

2. Trypan blue staining

In order to resist invasion of germs, most of plants prevent the spread of germs to surrounding cells through programmed cell death, as shown in fig. 19 and a, blue parts of leaves of StHB6 over-expressed plants after inoculation with late blight for 5 days are smaller than blue parts of WT leaves, and blue parts of leaves of StHB6 interference plants are larger than blue parts of WT leaves, and as a result, as shown in fig. 19 and b, the diameter of lesions of leaves of StHB6 over-expressed plants after trypan blue staining is significantly reduced compared with WT, and the diameter of lesions of leaves of StHB6 interference plants after trypan blue staining is significantly increased compared with WT. And verifying the disease spot size result after inoculating the pathogenic bacteria of the late blight again, wherein StHB6 participates in resistance regulation of the late blight and is positively regulated.

Example 9 regulatory network analysis of tHB6 transcription factors

1. StHB 6-binding StTCTP promoter

After earlier laboratory studies with tobacco NtHB6 and review of the relevant literature, it was found that the translational control tumor protein (Translationally controlled tumor protein, TCTP), also known as P23 in the human genome, is a protein that might assist in the transport of PVY in solanaceous plants. As shown in FIG. 20, the promoter of the potato TCTP gene was analyzed, and found to have the binding motif [ TAATNATT ] of Arabidopsis AtHB6 in the promoter of StTCTP.

To confirm the binding of transcription factor StHB6 to the promoter of StTCTP, we performed a dual luciferase reporter gene experiment. The gDNA of E3 is used as a template, a TCTP-1 (Soltu. DM.01G 039490.1) promoter sequence shown in SEQ ID No.24 and a TCTP-2 (Soltu. DM.01G 039500.1) promoter sequence shown in SEQ ID No.25 which are included in [ TAATAATT ] cis-acting elements are amplified by PCR, and constructed on a vector, then pGreenII 0800-LUC vector and empty pCAMBIA1300-eGFP vector, pGreenII 0800-LUC-TCTP vector and empty pCAMBIA1300-eGFP are respectively subjected to heat shock to be transferred into agrobacterium GV3101, and then respectively injected into Nicotiana benthamiana together, fluorescence is observed and sampled after 2 days, and liquid nitrogen quick freezing is performed.

Fluorescence values of LUC and REN were detected and the LUC/REN ratio was calculated. The results are shown in fig. 21, which shows that the LUC/REN ratio co-injected into nicotiana benthamiana is significantly lower than the control, indicating that StHB6 interacts with the StTCTP promoter and acts as an inhibitor.

2. Relationship among StHB6, PVY-CP and StTCTP

In summary, stHB6 binds to the StTCTP promoter and significantly inhibits transcription of StTCTP, and it is speculated that StHB6 positively regulates PVY resistance in plants by inhibiting StTCTP transcription such that TCTP cannot participate in PVY transport.

By detecting the expression of StHB6, stTCTP and PVY-CP in the inoculated leaves and upper leaves at 4 days, 11 days and 21 days after PVY infection, as shown in FIGS. 22, A to D, the relative expression amount of StHB6 in each transgenic plant was measured in the inoculated leaves and upper leaves after 4 days, 11 days and 21 days after PVY infection of each transgenic plant of StHB6. FIG. 22, F shows that the expression level of StHB6 in the upper leaves of the 4 days of the over-expressed plants is increased, the expression levels of PVY-CP and StTCTP are significantly reduced, and the expression level of StHB6 in the upper leaves of the interference lines is reduced so that the expression levels of PVY-CP and StTCTP are significantly higher than that of WT; as shown in fig. 22, g, the expression level of PVY-CP in the over-expressed strain was significantly reduced at day 11 of inoculation with PVY virus; as shown in FIG. 22, H, the relative expression level of PVY-CP in the StHB6 over-expressed strain was significantly reduced compared to WT at day 21 of PVY virus inoculation, whereas the relative expression level of PVY-CP in the StHB6 interfering strain was significantly increased compared to WT, as shown in FIG. 22, L, there was no significant difference in StTCTP expression compared to WT, indicating that StTCTP may be expressed well in pre-inoculation of PVY, and later TCTP expression was not significantly expressed, demonstrating that StTCTP expression was induced by potato virus in early stages of potato infection. In conclusion, the relative expression amounts of PVY-CP and StTCTP after 4 days, 11 days and 21 days of PVY inoculation of transgenic plants and interference of the overexpression of StHB6 are detected through RT-qPCR, and the assumption is made that StHB6 can not participate in PVY transportation by inhibiting the transcription of StTCTP, so that the transmission and accumulation speed of PVY viruses are delayed, and the PVY resistance of plants is positively regulated.

The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims

2. The use according to claim 1, characterized in that: the amino acid sequence of the transcription factor StHB6 is shown as SEQ ID NO. 22.

3. The use according to claim 1, characterized in that: the nucleotide sequence of the transcription factor StHB6 is shown as SEQ ID NO. 21.

4. The use according to claim 1, characterized in that: the application of the overexpression transcription factor StHB6 in improving the single plant tuber number of the potatoes.

5. The use according to claim 1, characterized in that: use of overexpressing transcription factor StHB6 in increasing the potato yield of potatoes.

6. The use according to claim 1, characterized in that: use of overexpressing the transcription factor StHB6 for increasing the total potato weight of potatoes.

7. The use according to claim 1, characterized in that: use of overexpressing the transcription factor StHB6 for increasing the average potato weight of potatoes.

8. The use according to claim 1, characterized in that: use of overexpressing the transcription factor StHB6 for increasing HPV resistance of potato.

9. The use according to claim 1, characterized in that: use of overexpressing the transcription factor StHB6 for increasing late blight resistance of potato.

10. The use according to claim 1, characterized in that: the method for over-expressing the transcription factor StHB6 comprises the steps of connecting the nucleotide sequence of the StHB6 in claim 3 with pRC26B vector skeleton through EcoR I and SpeI cleavage sites to obtain a recombinant expression vector, and then transforming potatoes under the mediation of agrobacterium to obtain a transgenic plant over-expressing the transcription factor StHB6.