Method for identifying resistance function of aphid-resisting genes through transforming hairy roots of soybean

A technology of aphid-resistant gene and hairy root, which is applied in the field of identifying the resistance function of the aphid-resistant gene by transforming soybean hairy root, can solve problems such as difficult control of aphids, and achieve simple and convenient implementation, accurate identification, convenient and reliable identification Effect

Inactive Publication Date: 2013-10-09

SHANGHAI JIAO TONG UNIV

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AI-Extracted Technical Summary

Problems solved by technology

According to the literature, soybean aphids are specialized and overwinter in the roots of several Rhamna plants; the identification of soybean aphid resistan...

Method used

[0044] Functional gene identification results: the average relative growth rate and the average relative reproduction rate of aphids were not much different between the non-transformed aphid-resistant genome and the control group. The number of aphids on the hairy roots with the aphid-resistant gene function obtained through screening was 4.3 times that of the original, while the number of aphids on the hairy roots without the aphid-resistant gene was 28 t...

Abstract

The invention discloses a method for identifying the resistance function of aphid-resisting genes by transforming hairy roots of soybean and relates to the field of methods for crop tissue culture and transgenosis. According to the method, the hairy roots are cultured by infecting mature seeds of the soybean by agrobacterium rhizogenes, and the effect of the aphid-resisting genes to resist against soybean aphids is verified through the transgenic hairy roots. The method mainly includes a cotyledon obtaining and culturing technique for the mature seeds of the soybean, an infection transformation technique for the agrobacterium rhizogenes, a degerming and continuous culturing technique for the transgenic hairy roots, a transgenic hairy root aphid inoculation technique, and an identification technique for the aphid-resisting functional genes of the hairy roots. The method is simple, easy to operate, free of limitation of seasons and capable of effectively and quickly identifying the effect of the target genes to resist against the soybean aphids through the transgenic hairy roots and providing reference for resistance identification of other crops.

Application Domain

Horticulture methodsPlant tissue culture +1

Technology Topic

ApidaeCrop +8

Examples

Experimental program(1)

Example Embodiment

[0023] Example 1

[0024] The soybean receptor was selected from the aphid-susceptible variety Williams82, using soybean hairy roots to identify the function of Rag1 aphid resistance gene, the steps are as follows:

[0025] 1) Obtain soybean cotyledons: select healthy and disease-free mature soybean seeds, rinse with water, sterilize with 75% alcohol for 30 seconds, and 0.1% Ca(ClO) 2 Sterilize for 20 minutes, shake about 5 times during the period, wash away Ca(ClO) with sterile water 2 Residue, rinse 5 times, inoculate in germination medium, light 16h/day, 25±3℃; the composition of germination medium is: MSB+7g/L agar+30g/L sucrose, pH value is 5.8; The cotyledons are the cotyledons formed after 5 days of sterile germination of mature soybean seeds.

[0026] 2) Agrobacterium rhizogenes infection and transformation to induce hairy roots: Use a sterile scalpel to cut the cotyledon from the hypocotyl, and cut a wound with a radius of about 4mm on the cotyledon near the hypocotyl and the abaxial surface. The wound is as deep as The central axis (but not through the cotyledons). Place the cut cotyledons in a petri dish with filter paper, and add 1/4 MSB5 liquid medium (to make the filter paper completely wet but no residue), and add 20 μL of three bacterial liquids to the wound of the cotyledons, respectively : Agrobacterium rhizogenes K599 (Rag1-pHB-K599) with target gene Rag1, K599 strain (pHB-K599) with empty vector pHB and K599 strain (K599) without pHB vector, seal the petri dish with a sealing film After 3 days of dark culture, the light culture was resumed, and the light was 16h/day, 28℃, and cultured for 1 week. The wound began to appear callus, followed by hairy roots.

[0027] The construction method of the strain Rag1-pHB-K599 carrying the resistance gene Rag1 for inducing hairy roots is as follows: amplify the target gene fragment by RT-PCR with specific primers, transform E. coli DH5α, and extract the plasmid for double enzyme digestion after verification by sequencing , Connect the digested target gene fragment to the vector pHB recovered by the same digestion, and then transform the recombinant plasmid Rag1-pHB into Agrobacterium K599, on the YEB plate containing streptomycin (Str) and kanamycin (Kan) Screening and PCR verification on the above, and then expand the culture to OD 600 =0.6~0.8, the infected cotyledon wound is genetically transformed, and dark culture induces hairy roots. (The relevant operations involved in the experiment, such as competent preparation, gel recovery, transformation connection, etc., are strictly in accordance with the "Molecular Cloning Experiment Guide" 》The third edition and relevant kit instructions).

[0028] 3) Sterilization and continued cultivation of hairy roots: Cut the hairy roots cultivated to 5 cm, place them in a sterile culture flask, and rinse with sterile water containing Cef500mg/L for 3 times. More preferably, the culture medium for continued culture after sterilization is 1/4MSB5+Cef500mg/L, and the root tips of hairy roots are submerged in the culture medium with sterile filter paper and cultured for 3 days.

[0029] 4) Inoculation of aphids on hairy roots: select three groups of hairy roots (Rag1-pHB-K599, pHB-K599, K599) that grow basically the same, and use a brush to homogenize the aphid-susceptible soybeans on the isolated leaves The third-instar soybean aphids were inoculated on the hairy roots of each group, and 3 aphids were received in each petri dish.

[0030] 5) Identification of the anti-aphid function of the target gene in the transgenic hairy roots: 14 days after the inoculation of the aphid, the number of aphids, the aphid reproduction rate and other parameters are counted. The average relative growth rate and average relative reproduction rate of soybean aphids were used as the basis for the identification of target gene aphid resistance, among which,

[0031] The calculation formula of the average relative growth rate (g/d) is:

[0032] Average relative growth rate = [LN (total weight of final aphids)-LN (total weight of initial aphids)]/14×100%

[0033] The average relative growth rate of transgenic aphid-resistant gene (Rag1-pHB-K599)

[0034] =[LN(0.0034)-LN(0.0008)]/14×100%=10.34%

[0035] Average relative growth rate of non-transformed aphid-resistant genes (pHB-K599)

[0036] =[LN(0.0219)-LN(0.0008)]/14×100%=23.64%

[0037] The average relative growth rate of the control group (K599)

[0038] =[LN(0.0227)-LN(0.0008)]/14×100%=23.89%

[0039] The calculation formula of the average relative reproduction rate (No./d) is:

[0040] Average relative reproduction rate = (final aphids number-initial aphids number)/14

[0041] The average relative reproduction rate of transgenic aphid-resistant genes (Rag1-pHB-K599)=(13-3)/14=0.714

[0042] The average relative reproduction rate of non-transformed aphid-resistant genes (pHB-K599)=(84-3)/14=5.78

[0043] The average relative reproduction rate of the control group (K599)=(87-3)/14=6.00

[0044] Functional gene identification results: the average relative growth rate and average relative reproduction rate of aphids are not much different in the non-transformed aphid-resistant genome group and the control group. The number of aphids on hairy roots screened with aphid resistance gene function was 4.3 times that of the original, while the number of aphids on hairy roots with non-transformed aphid-resistant genes was 28 times the original number; The average relative growth rate and average relative reproduction rate of Aphid were 2.3 times and 8.1 times that of transgenic aphid-resistant genes. The resistance function of aphid-resistant genes on hairy roots was significantly different. The method of identifying the aphid resistance gene function through soybean hairy roots can quickly and accurately identify the aphid resistance gene function.

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