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Studies on the Transformation of Na~+/H~+ Antiport Gene SbNHX1 into S. Guianensis Cv. Reyan No.5

Author: CaiJie
Tutor: HuXinWen；GuoJianChun
School: Hainan University
Course: Biochemistry and Molecular Biology
Keywords: Stylosanthes guianensis cv. Reyan No.5 transgenic plant salt tolerance PCR-Southern transplanting
CLC: S541.9
Type: Master's thesis
Year: 2011
Downloads: 42
Quote: 0
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Abstract


Stylosanthes guianensis cv. is an excellent tropical legume forage, which has the characteristics of drought-resistance, infertile-resistance, high yield of stem and leaf, good quality, stronge-re si stance of acid red loam; however it shows poor salt-tolerance. In order to increase salt tolerance and acquire the newly characteristic of salt resistance species, a salt-tolerant related gene was transformed to the genome of S. guianensis cv. Reyan No.5. The researches in this study include the establishment of S. guianensis in vitro culture system; the condition optimization for explant transformation; the transformation of the salt-resistant Na+/H+ antiporter gene SbNHXl isolated from the halophyte Salicornia bigelovii Terr, to the genome of S. guianensis cv. Reyan No.5 through Agrobacterium LBA 4404; molecule detection in parts of transgenic plants by PCR, RT-PCR and PCR-Southern. The results as the following:1) The best method for sterilization of S. guianensis cv. Reyan No.5 is as the following:the seeds were treated by 80℃,5 min;75% ethanol for 45 s, then washed 3-5 times with fresh water; 0.1% HgCl2for 5 min and washed 3-5 times with fresh water. The sterilization ratio was up to 92%.2) The best callus induction medium of S. guianensis cv. Reyan No.5 is MS+1 mg/L NAA+4 mg/L 6-BA +30 g/L sucrose+8.5 g/L agar powder, pH 6.0-6.3. The bud differentiation medium is 0.1 mg/L NAA+MS+0.4 mg/L 6-BA+30 g/L sucrose+8.5 g/L Agar powder, pH 6.0-6.3. The root inducible medium is MS solid medium or 1/2 MS solid medium.The best explant is the cotyledon.3) For gene transformation to S. guianensis cv. Reyan No.5, the optimal concentration of Car and Hyg is 300 mg/L and 15 mg/L;the submersion time of cotyledon with Agrobacterium is 10～15 mins; co-culture time is 60 h and co-culture temperature is 25℃.4) Under the above transformation system, Na+/H+ antiport protein gene SbNHX1 from the halophyte Salicornia bigelovii Terr. has been transformed to the genome of S. guianensis cv. Reyan No.5 through Agrobacterium LBA 4404 and obtained 205 transgenic plants with antibiotic-resistance. Parts of these transformants have been identified by PCR, PCR-Southern and RT-PCR, there is 10 indepent transgenic lines were identified in transcription mRNA level. Thus, the Na+/H+ antiporter gene SbNHX1 has been transformed to the genome of Stylosanthes guianensis cv. Reyan No.5. 5) The best transplanting substance is red soil+decay soil+sand (2:1:2) and best transplanting period is Feb.-Mar. during a year. By this method we have successfully transplanted 205 transgenic plants into substance.

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