CN105219794A - A kind of photosensitizing effect that utilizes formulates the method for keeping away shade corn germplasm - Google Patents

Abstract

The invention relates to a method for transforming corn calluses and immature embryos by using phytochrome genes to create shade-avoiding corn germplasm, which belongs to the field of plant transgenic technology and crop genetic breeding. The invention comprises the following steps: constructing a plant expression vector of the phytochrome gene; transforming the corn callus and immature embryo by the plant expression vector through the method mediated by Agrobacterium; performing PCR detection on the regenerated plant after transformation to determine the positive plant; obtaining the positive Plants are self-bred and reserved to obtain stable high-generation transgenic plants; planting schemes with high and low densities are designed according to random block experiments, planting phytochrome gene plants and their hybrid combinations with commonly used inbred lines, and statistical analysis of plant type traits in the field ; Analyze the experimental results, and screen transgenic maize germplasm with excellent shade avoidance and yield. The invention utilizes the influence of over-expression of corn endogenous phytochrome gene on its plant type, plays a role in corn plant type breeding with corn transgenic technology, and cultivates corn varieties that can be reasonably densely planted.

Description

A method for creating shade-avoiding maize germplasm using light-sensitive genes

technical field

The invention belongs to the field of plant transgenic technology and crop genetic breeding, and specifically relates to a method for creating shade-avoiding maize germplasm by using a light-sensitive gene.

Background technique

With the decrease of arable land, people's demand for corn is increasing, and increasing the yield of corn is the main production goal of our country. Breeding excellent maize germplasm is the key to increasing maize yield, and transgenic breeding technology is one of the more economical and effective measures compared with conventional breeding methods.

Among the various factors affecting maize yield, light signal is particularly important. Light is not only an energy source, but also an important environmental signal. Phytochromes play an important role as the main photoreceptors in the process of plant ontogeny. In plants, phytochrome usually constitutes a gene family of several homologous genes, each species has its own unique gene family, and its function also has some special changes.

From the research on phytochrome, it can be seen that phytochrome has a direct impact on the yield of crops. Such as: Alexandra et al. used the Arabidopsis PhyB gene to transform potatoes, and finally increased the potato tuber yield. Boccalandro et al. also used the Arabidopsis PhyB gene to transform potatoes. The results of planting the transgenic plants in the field showed that they could increase light and efficiency, and the negative negative response was weak when they were highly densely planted. After Kong et al. transferred Arabidopsis PhyA gene into rice, the yield of rice increased and the agronomic traits of rice were improved. But so far, there is no relevant report about using phytochrome gene to play a role in maize plant type breeding and cultivating maize varieties suitable for reasonable dense planting.

The invention utilizes the phytochrome signal pathway to transform the phytochrome properties of the corn, transfers the phytochrome gene in the corn into the corn, improves the shade avoidance of the corn, improves the quality and characteristics of the corn, and further increases its yield. In addition, the present invention selects the endogenous phytochrome gene of maize for research, and the reason is that so far, the crop traits are basically modified by transferring exogenous phytochrome genes, such as transferring the phytochrome of Arabidopsis thaliana into Rice, wheat, potato, cotton, etc. There is no research on the transfer of endogenous phytochrome genes. The transfer of foreign genes into the recipient may cause gene rejection, making it difficult for the gene to be transferred, and even if the transfer is successful, it may cause gene silencing. However, the transfer of endogenous genes greatly increases the positive rate of transgenic lines and reduces the possibility of gene silencing. Therefore, the present invention has greater value and significance to the development of maize germplasm and the improvement of maize shade avoidance.

Contents of the invention

The main purpose of the present invention is to solve the problem of corn shade avoidance, and provide a new way of utilizing phytochrome gene overexpression in corn to cause beneficial changes in plant light signal mechanism, thereby improving the plant type and shade avoidance of plants.

The method for creating shade-avoiding maize germplasm provided by the present invention includes introducing PhyA1 , PhyA2 , and PhyB1 genes in the phytochrome gene family into corn respectively, and after obtaining transgenic maize with overexpression of phytochrome genes, performing field test screening and creating shady-avoiding maize germplasm. Steps for shade-excellent maize germplasm.

In order to realize the foregoing invention object, the technical scheme that the present invention adopts is as follows:

(1) Construction of plant gene expression vector:

1) Acquisition of the target gene: amplify the endogenous phytochrome genes PhyA1 , PhyA2 , and PhyB1 from corn B73 respectively; after sending to the company for sequencing, store the target gene product with the correct amplified sequence in a 4°C refrigerator for later use;

2) Obtaining large vector fragments:

Plant expression vector large fragment (pTF101.1 -Ubi-T-nos ):

On the pTF101.1 basic vector (which contains the Bar gene as a screening gene), a new expression vector is constructed after adding the promoter Ubiquitin and the terminator T-NOS, and the phytochrome genes ( PhyA1 , PhyA2 , PhyB1 ) will be inserted in pTF101. 1- Between the BamHI and SmaI restriction sites in the T-DNA region of the Ubi - T-nos vector, the maize ubiquitin promoter Ubiquitin is used to initiate the expression of the target gene, and the T-nos terminator is used to terminate the expression of the target gene;

Plant expression vector large fragment (pCUbi1390):

Its T-DNA region contains the hygromycin phosphotransferase ( HPT ) selection marker gene and the CaMV35S promoter that starts the hygromycin phosphotransferase ( HPT ), and the maize phytochrome genes ( PhyA1 , PhyA2 , PhyB1 ) will be inserted in T - Between the Bam HI and Spe I restriction sites in the DNA region, the maize ubiquitin promoter Ubiquitin is used to initiate the expression of the target gene, and the T-nos terminator is used to terminate the expression of the target gene;

3) After preparing the above DNA fragments, digest the DNA fragments of PhyA1 , PhyA2 , PhyB1 and the large carrier fragments respectively, and connect them overnight at 16°C, and send the ligation products to the company for sequencing after verification by enzyme digestion; the sequencing results are correct Transform the competent cells of Agrobacterium by heat shock of the connection product, screen the transformed Agrobacterium with spectinin and rifamycin resistance medium, pick a single colony for bacterial liquid PCR identification; save the verified Agrobacterium liquid for future use ;

(2) Transformation of corn callus by Agrobacterium-mediated method:

1) Dip dyeing:

Inoculate the Agrobacterium containing the constructed plant expression vector into the YEP medium containing 50mg/L rifampicin and 50mg/L spectinomycin, and culture it at 28°C for 2 days; pick a single colony in 1mL YEP medium containing the corresponding antibiotic 28°C, 180r/min shaking culture for 12h, then add it to 50mL medium containing corresponding antibiotics to expand the culture to OD ₆₀₀ =0.5; 4°C, 4000r/min centrifuge for 10min to collect the bacteria, and use an equal volume of liquid to culture Submerge the callus of the excellent maize inbred line to be transformed into the dipping culture solution containing acetosyringone (AS), and dip in the dark for 3 minutes;

2) Co-cultivation, recovery and screening:

Put the callus soaked with Agrobacterium on a petri dish containing sterilized paper, blot the excess water, empty the dish and cultivate in the dark at 21°C for 3 days; inoculate the callus after 3 days on the recovery medium at 25°C Dark culture for 7 days; callus after recovery culture was transferred to selection medium containing 1.5 mg/L herbicide glufosinate; 25 days later, resistant callus was transferred to fresh selection medium; continuous selection For 3 generations, the herbicide concentration is increased by 1.5mg/L, 3mg/L, and 4.5mg/L; each transfer will eliminate the browned callus;

3) Regeneration of transgenic plants:

Select the resistant callus and transfer it to the differentiation medium. After 2-3 weeks, transfer the differentiated seedlings to the rooting and strong seedling medium. The rooting and strong seedling medium is: MS salt and vitamin + inositol 100mg/ L+ rooting powder ABT0.25g/L+ sucrose 30g/L+ agar 6g/L, pH6.0; culture at 26°C under light for 16 hours/d to obtain T ₀ transgenic regenerated plants; carry out molecular tests such as PCR detection and sequencing on the regenerated plants, Positive T ₀ plants were screened out and kept for selfing;

(3) Design high and low density planting schemes according to random block experiments, plant transgenic plants and their hybrid combinations with commonly used inbred lines (3 to 5), investigate and measure relevant data, analyze field test results, and obtain avoidance by screening. Transgenic maize germplasm with excellent shade and yield.

So far, the present invention has successfully obtained high-generation transgenic maize plants, and field experiments have proved that overexpression of phytochrome genes has a significant impact on maize plant type.

Compared with prior art, the beneficial effect of the present invention is:

So far, there have been no related reports on the use of phytochrome genes to play a role in maize plant type breeding. Maize phytochrome genes were transferred into maize, leading to overexpression, supporting the hypothesis that overexpression of phytochrome plays an important role in important agricultural crops. In recent years, biotechnological approaches have attempted to reduce the shade-avoidance response to increase the yield of high-density crops. An effective strategy is to suppress the elongation response through the overexpression of phytochromes. Because phytochrome controls the physiological process of plants, the overexpression of phytochrome gene affects the regulation mechanism of plant physiological process.

In addition, the present invention selects the endogenous gene of maize for research. So far, the existing research is basically to modify the crop traits by transferring the exogenous phytochrome gene, such as transferring the phytochrome of Arabidopsis into rice, wheat, potato, etc. , cotton and so on. There is no research on the transfer of endogenous phytochrome genes. The transfer of foreign genes into the recipient may cause gene rejection, making it difficult for the gene to be transferred, and even if the transfer is successful, it may cause gene silencing. However, the transfer of endogenous genes greatly increases the positive rate of transgenic lines and reduces the possibility of gene silencing. Through the Agrobacterium-mediated transformation of maize in transgenic technology, the quality and characteristics of maize are improved, and maize transgenic plants with excellent shade avoidance are bred, and they play a role in maize plant type breeding, fundamentally solving the limitation of maize planting density Problems, significantly increase corn yield, improve its economic value.

Description of drawings

Fig. 1 is the electrophoresis diagram of amplifying target gene PhyA1 , PhyA2 ;

Fig. 2 is the electrophoresis diagram of the amplified target gene PhyB1 ;

Fig. 3 is a callus diagram induced by immature embryos of corn;

Fig. 4 is the differentiation figure of corn callus;

Fig. 5 is the seedling transgenic root culture diagram that Fig. differentiates;

Figure 6 is a control difference diagram of transgenic lines at the jointing stage, the left side is the transgenic 18-599R:: ZmPhyA1 line, and the right side is the transgenic 18-599R:: ZmPhyB1 line;

Fig. 7 is a control difference diagram of the transgenic lines at the tasseling stage.

detailed description

The above content of the invention of the present invention will be further described in detail below in conjunction with specific embodiments.

However, it should not be construed that the scope of the above-mentioned subject matter of the present invention is limited to the following examples. Without departing from the above-mentioned technical idea of the present invention, various replacements and changes made according to common technical knowledge and customary means in this field shall be included in the scope of the present invention.

Example 1

This embodiment is the cloning of phytochrome genes PhyA1 , PhyA2 , PhyB1 :

1. Cloning of PhyA1 and PhyA2 :

PCR primers were designed with PrimerPrimier5.0 software, and the synthesized full-length primers were diluted to a concentration of 10 μmol, and the reverse transcription product cDNA was subjected to PCR with LATaq enzyme, and the annealing temperature was 62°C.

A1F: 5'TCGGATCCGAATTCATGTCTTCCTTGAGGCC3',

A1R: 5'GTCGAGACTAGTTCAATGTCCAGCTGCTGA3';

A2F: 5'TCGGTACCGGATCCGAATTCATGTCTTCCTCGAG3',

A2R: 5'GTCGAGACTAGTTCATCGTCCAACTGCTGA3'.

The amplification system (20μL) is:

After the reaction, the PCR product was detected on 1.0% agarose (Figure 1), and the target band was recovered and sent to the company for sequencing. The result was correct.

2. Cloning of PhyB1

B1F: 5'CGC GGATCC ATGGCGTCGGGCAGCCGCG3'

B1R: 5' TCC CCCGGG TCAGACGATTTCTCTACCAGCTGCTGGACGA3'

A BamHI restriction site (underlined base) is added to the upstream primer, and a Sma I restriction site (underlined base) is added to the downstream primer. Add 3 protective bases before the restriction site.

The amplification system (50μL) is:

5×psBuffer10μL

dNTP mix 4 μL

Upstream primer 2 μL

Downstream primer 2 μL

DNA template 5 μL

High Fidelity Enzyme 0.6 μL

ddH2O26.4μL

The amplification procedure is:

94°C, 3min; 98°C, 10s; 72°C, 90s; 34 cycles; 72°C, 8min; 12°C hold

After the reaction, the PCR product was detected on 1.0% agarose (Figure 2), and the target band was recovered and sent to the company for sequencing. The result was correct.

Example 2

This embodiment is the transformation process of corn by Agrobacterium-mediated method:

1. Transformation of Agrobacterium heat-shock competent cells

(1) Take 100 μL of Agrobacterium competent cells stored at -70°C (preserved in the laboratory), and place them on ice to thaw to the state of ice-water mixture.

(2) Add 1 μL of the constructed plant expression vector, shake gently, and place on ice for 30 minutes.

(3) Quick-freeze in liquid nitrogen for 1 min, bathe in water at 37°C for 5 min, then add 1mL YEB liquid medium (containing 50mg/mL rifampicin), and culture with slow shaking at 28°C for 4h.

(4) Spread 100 μL of the culture on a YEB plate containing rifampicin (50 mg/mL) and spectinomycin (50 mg/mL), place it upside down for 30 min, and invert the culture dish after the bacterial solution is completely absorbed by the medium. Incubate at 28°C for about 48h.

(5) Pick a single clone in an EP tube of YEB liquid medium containing rifampicin (50mg/mL) and spectinomyces (50mg/mL), shake on a shaker at 180r/min for 4-5h at 28°C, and perform PCR detection , and the Agrobacterium liquid to be detected as positive was stored in a 4°C refrigerator for later use.

2. Diffusion Transformation of Agrobacterium

The liquid was prepared according to the formula of the liquid culture liquid in the experimental material, and sterilized by autoclaving at 115°C for 20 minutes. Store in a refrigerator at 4°C.

Transfer the positive Agrobacterium liquid detected by PCR to the resistant YEB liquid culture containing rifampicin (50mg/mL) and spectinomycin (50mg/mL), culture in dark at 28°C with 200r/min shaking until OD ₆₀₀ = Around 0.6. Aliquot the bacterial solution into 50mL centrifuge tubes, centrifuge at 4000r/min for 10min to collect the bacterial cells. Then add the liquid to the centrifuge tube containing the bacteria to mix the bacteria, and pour it into the corn callus to be soaked.

(1) Dipping callus

The material of about 12 days after pollination of the excellent inbred line maize was selected, and the callus used in the experiment of immature embryo induction with a length of about 1.0 mm was picked. The process is as follows: After taking the materials about 12 days after pollination back to the laboratory, peel off the leaves of corn materials that are intact and free from insect bites, and sterilize them with 75% alcohol continuously during the peeling process. Pick the embryos of the peeled corn in the ultra-clean workbench. First, use a blade that has been autoclaved and burned red three times on the flame to cut off half of the kernel of the corn, just to the depth of the immature embryo, and use sterile tweezers that have been autoclaved and burned red three times on the flame The young maize embryos were picked, and the scutellum was placed on the induction medium for callus induction. Cultivate in the dark at 28°C for about 7 days, remove the adventitious buds in time to induce callus, and replace the induction medium after about 15 days and culture for another 15 days, during which the adventitious buds are removed in time. Corn callus can be induced in about 30 days (Figure 3), which has the characteristics of light yellow surface color, loose and granular shape. The induced callus was transferred to the subculture medium for subculture, and the subculture medium was replaced once at 28° C. for dark culture for 15-20 days. A total of 3 generations.

Add the callus of the third subculture for about 7-10 days into a sterilized vial with sterile tweezers, pour the above-mentioned liquid containing Agrobacterium cells into the small bottle, and pour the liquid into the vial after dipping for 3 minutes. After 30-45 minutes, the callus was transferred to a petri dish with filter paper to absorb the residual bacterial liquid on the surface. After 30-45 min, it was transferred to an empty dish culture medium and cultured in the dark at 21°C for 3 days. After 3 days, the calli were transferred to the recovery medium and cultured in the dark at 28°C for 7 days.

(2) Dip dyeing immature embryos

The corn of the excellent inbred line with about 12 days of pollination and about 1mm of immature embryo was selected for embryo picking. The method is similar to previous work on maize callus infection. First tear off the bract leaves of the corn layer by layer, and spray 75% disinfectant alcohol continuously while tearing off. When the remaining 3 layers of bracts are left, the filaments of the corn are cut off, and then the last bracts are peeled off and put into the ultra-clean workbench. Use a sterilized razor blade to cut off half of the corn kernels that just exposed the immature embryos, then pick out the immature embryos with sterilized tweezers, and put them into EP tubes filled with 1.5mL dipping solution, about 100 per tube. Young embryos. After washing twice with the dipping solution, the immature embryos were dipped for 5 minutes with the dipping solution containing Agrobacterium. Pour it into the co-cultivation medium, blot dry with sterile filter paper, and culture in the dark at 21°C for 3 days. The immature embryos were transferred to recovery medium and cultured in the dark at 28°C for 7 days. During this period, the adventitious buds should be clipped in time.

(3) Screening of resistant callus and plant regeneration

Inoculate the callus that has been cultured for 7 days on the screening medium containing herbicides. According to the previous experimental experience, the gradient concentration of herbicides should be selected as 1.5, 3 and 4.5mg/L in order to prepare The screening medium was cultured in the dark at 28°C, and each concentration was screened and cultured for 20-25 days. During this period, the callus showed obvious browning and death. After 3 times of screening at different concentrations, the non-browning callus was transferred to the differentiation medium for differentiation culture (Figure 4), and adventitious shoots and roots were removed, and the condition was dark culture at 28°C for 10 to 15 days.

The resistant callus was transferred to the differentiation medium for light culture at 28°C. When the small shoots of 4-5 cm are differentiated, they are transferred to the medium for rooting and strong seedlings for rooting culture (Figure 5). After the shoots on the rooting medium grew 2-3 main roots, the bottle cap was opened and cultivated for 1-2d. After the seedlings adapt to the indoor environment, carefully take them out of the culture bottle, wash the root medium with sterile water, cut off the old roots and dead leaves, and transplant them into nutrient soil sterilized by high temperature. After transplanting and surviving, wait for the seedlings to grow strong and then transplant them into the field for cultivation.

Example 3

This embodiment is the field phenotype identification of stable positive plants:

The transgenic lines and the control inbred lines were planted at two or three densities according to the comparative design, and repeated several times.

ZmPhyA1 , ZmPhyA2 , ZmPhyB1 and the cross combinations with commonly used inbred lines were planted in different densities according to random block design.

Field management was carried out according to the local conventional cultivation mode (Fig. 6 and Fig. 7). After the tasseling stage, the flowering stage, plant height, ear height, leaf length, leaf width and leaf angle of the next leaf after the tassel stage were investigated. Analysis of variance and multiple comparisons were performed on the measured data with ExcelWPS and DPS statistical analysis software. Use ExcelWPS software to analyze the data, and calculate the average value of various data in each district under investigation. The data of each plot was compared with the data of adjacent control plots. Statistical analysis of phenotypic data was carried out by using ExcelWPS and DPS statistical analysis software, and significant difference analysis was carried out by LSD method. Calculate the average value of various data of each plot in the survey, input it into the DPS software, and conduct parameter estimation and normality test on each data. When the data conforms to the normal distribution, it conducts variance analysis of random block two-factor experiment. Select the LSD method for the multiple comparison method, click OK to form the variance analysis table, and then obtain the significant level P value, analyze the block effect of various data, the density factor and the strain factor and the interaction between the two. When the significant level P value in the analysis of variance table was less than or equal to 0.05, further multiple comparison analysis was performed. Multiple comparisons can be used to further examine the differences between pairs of treatments. It can analyze multiple comparisons between each strain and multiple comparisons between different density treatments of each strain.

The data so far show that this research method has a significant impact on the change of maize plant type. Transplantation of 18-599R::ZmPhyA1 gene in maize makes the leaves of the plant become slender, the angle between the leaves becomes smaller, and the grain becomes smaller; :: ZmPhyB1 gene leaves become shorter and wider, the whole plant is denser, and the seeds become larger. These two kinds of transgenic maize showed that the leaf angle was significantly reduced at the same time, and they were suitable for reasonable dense planting.

Claims (3)

1. utilize phytochrome gene to formulate a method of keeping away shade corn germplasm, it is characterized in that comprising the following steps:

Plant the structure of expression vector:

1) acquisition of goal gene: amplification obtains the endogenous phytochrome gene of corn respectively from corn B73 phyA1, phyA2, phyB1; After sending company to check order, goal gene product correct for extension increasing sequence is kept at 4 DEG C of refrigerators for subsequent use;

2) acquisition of carrier large fragment:

Plant expression vector large fragment (pTF101.1- ubi-T-nos):

PTF101.1 carrier is carrier (wherein contains bargene is as screening-gene), add promotor ubiquitin, the new expression vector that builds after terminator T-NOS, phytochrome gene ( phyA1, phyA2, phyB1) will pTF101.1-be inserted in ubi- t-nosin carrier T-DNA district bamHIwith smaIbetween restriction enzyme site, what start this destination gene expression is maize ubiquitin promotor ubiquitin, what stop this destination gene expression is t-nosterminator;

Plant expression vector large fragment (pCUbi1390):

In its T-DNA district containing hygromix phosphotransferase ( hPT) selectable marker gene and start hygromix phosphotransferase ( hPT) caMV35Spromotor, corn phytochrome gene ( phyA1, phyA2, phyB1) will be inserted in T-DNA district bamhI and spebetween I restriction enzyme site, what start destination gene expression is maize ubiquitin promotor ubiquitin, what stop destination gene expression is t-nosterminator;

3) after getting out above-mentioned DNA fragmentation, then will phyA1, phyA2, phyB1dNA fragmentation and carrier large fragment cut through enzyme respectively, spend the night in 16 DEG C of connections, product will be connected and check order through digestion verification Hou Song company; By heat-shock transformed for connection product correct for sequencing result Agrobacterium competent cell, the Agrobacterium transformed with grand element, the screening of rifomycin resistance culture base, picking list bacterium colony carries out bacterium liquid PCR to be identified; Agrobacterium bacterium liquid after checking is saved backup;

(2) by agriculture bacillus mediated method maize transformation callus:

1) contaminate:

By the Agrobacterium inoculation containing the plant expression vector built to the YEP substratum containing 50mg/L Rifampin and 50mg/L spectinomycin, cultivate 2 days in 28 DEG C; The single bacterium colony of picking in containing in corresponding antibiotic 1mLYEP substratum, 28 DEG C, 180r/min shaking culture 12h, then added containing corresponding antibiotic 50mL substratum enlarged culturing to OD ₆₀₀=0.5; 4 DEG C, 4000r/min centrifugal 10min collection thalline, contaminate substratum with isopyknic liquid and suspend; The callus of good inbred lines to be transformed is immersed respectively the dip-dye nutrient solution containing Syringylethanone (AS), dark dip-dye 3min;

2) training altogether, recovery and screening:

Be placed on the culture dish containing sterilizing paper by the callus contaminating Agrobacterium, after blotting excessive moisture, emptying ware is cultivated, 21 DEG C of light culture 3d; Callus after 3d is inoculated into 25 DEG C of light culture 7d on recovery media; Callus after renewal cultivation is transferred on the Selective agar medium containing 1.5mg/L weedicide grass fourth phosphine; Resistant calli is transferred in fresh screening culture medium after 25d; In step sizing 3 generation, herbicide concentration increases progressively by 1.5mg/L, 3mg/L, 4.5mg/L; The callus of brownization is all eliminated in each switching;

3) regeneration of transfer-gen plant:

Choose resistant calli, transfer on division culture medium, after 2-3 week, the seedling differentiated is transferred on Rooting and hardening-off culture base, Rooting and hardening-off culture base is: MS salt and VITAMIN+inositol 100mg/L+ root-inducing powder ABT0.25g/L+ sucrose 30g/L+ agar 6g/L, pH6.0; 26 DEG C of illumination cultivation, 16 hours/d, namely obtains T ₀transgenic regenerated plant; PCR detection is carried out to regeneration plant, order-checking equimolecular detects, filter out positive T ₀plant selfing is reserved seed for planting;

(3) by randomized block experiment design height different densities Plant plane, (3 to 5) cross combination of plantation transfer-gen plant and itself and conventional self-mating system, researching determining related data, analyze field test results, screening obtains and keeps away shade and the excellent transgenic corns kind matter of output.

2. a kind of phytochrome gene that utilizes according to claim 1 formulates the method for keeping away shade corn germplasm, and it is characterized in that, the phytochrome gene that carrier construction utilizes, is have selected in phytochrome gene family phyA1, phyA2, phyB1.

3. a kind of phytochrome gene that utilizes according to claim 1 formulates the method for shade corn germplasm of keeping away, and it is characterized in that, positive plant need obtain more stable height for transgenic positive plant by repeatedly selfing screening of reserving seed for planting.