Rice-stress-resistance-related hot shock protein gene OsHsp17.0, and encoding protein and application of rice-stress-resistance-related hot shock protein gene OsHsp17.0
A technology of heat shock protein and encoded protein, which is applied in the field of plant heat shock protein gene and its encoded protein, and can solve problems affecting rice growth and development, rice yield and quality, etc.
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Embodiment 1
[0024] Embodiment 1, the acquisition of OsHsp17.0 gene overexpression plant
[0025] In order to better clarify the function of the OsHsp17.0 gene, the applicant overexpressed it in rice, and verified the function of the OsHsp17.0 gene from the phenotype of the transgenic plants.
[0026] 1. Construction of OsHsp17.0 gene overexpression vector pCAMBIA1301M-OsHsp17.0
[0027] The pCAMBIA-1301-multi vector containing the CAMV35S promoter was digested with restriction endonucleases BamH I and Xba I, and primers P1: 5'-CGGGATCCCCACCCAACTCTTTCTTCTTCC-3' and P2: 5'-GCTCTAGACTTGACCTTGACAAACTCCC-3' (containing BamH I Recognition site GGATCC and Xba I recognition site TCTAGA) the OsHsp17.0 gene obtained by PCR amplification was ligated with T4 DNA ligase, the ligated product was transformed into Escherichia coli DH5α, and the kanamycin-resistant gene was screened on LB plate The colony was extracted and the plasmid was identified by double enzyme digestion to obtain the positive plant...
Embodiment 2
[0040] Example 2, Molecular detection of OsHsp17.0 gene overexpression transgenic rice
[0041] The molecular detection of the OsHsp17.0 gene overexpression transgenic rice of the present invention is carried out by the RT-PCR method.
[0042] Select resistant and transplanted surviving plants screened by hygromycin, cut 1-2 leaves from each plant, and use Trizol reagent extraction method of Invitrogen Company to extract the total RNA of rice with slight modification, and the total RNA is removed by DNase I of Fermentas Company After that, the first strand of cDNA was synthesized with the reverse transcription kit (Rever Tra Ace Kit) of Toyobo Company, and the steps were carried out according to the instructions. The rice Actin actin gene RAc 1 was used as an internal standard for RT-PCR analysis. Actin primer sequences are as follows:
[0043] RAc1 Fw 5'-CTTCAAACACCCCTGCTATG-3'
[0044] RAc1 Rv 5'-TCCATCAGGAAGCTCGTAG-3'
[0045] Select OsHSP17.0-specific fragments for det...
Embodiment 3
[0051] Example 3, OsHsp17.0 Gene Overexpression Transgenic Rice Response to Drought Treatment
[0052] The present invention selects three independent overexpression transgenic homozygous rice lines obtained in Example 2 for phenotype screening. Seeds of homozygous transgenic lines (No. 1, No. 3, No. 8) and wild-type Nipponbare seeds were germinated in the medium for 1 week, then transferred to 1 / 2 Hoagland liquid medium for another 2 weeks, and the seedlings were taken out of the culture medium and wiped with absorbent paper. Blot dry the water on the root surface, expose to the air (25±1°C, relative humidity 40±5%) to dry for 9.5h, and then transfer to 1 / 2 Hoagland liquid medium to restore culture for 10d.
[0053] After being exposed to air for 9.5 hours under drought treatment, such as image 3 As shown in B, most of the leaves of transgenic rice overexpressed with OsHsp17.0 gene and the control wild type were wilted, showing severe drought stress phenotype.
[0054] Exp...
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