KARAN, Ratna,ROY, Gautam Kumar,SINGLA-PAREEK, Sneh Lata
申请号：
ES09806540
公开号：
ES2668998T3
申请日:
2009.08.10
申请国别(地区):
ES
年份:
2018
代理人:
摘要：
A procedure for the isolation of the hybrid histidine kinase gene - OsHK3b from IR64 indica rice for use in improving tolerance to salinity stress and drought stress of crop plants by overexpression of OsHK3b, characterized by (i) treatment of isolated cDNA from IR64 seedlings with OsHk3bF direct primer and OsHk3bR reverse primer by polymerase chain reaction (PCR) to produce the isolated amplified hybrid type histidine kinase gene - OsHK3b, (ii) cloning of the isolated gene - OsHK3b in the TOPO-TA2.1 vector to produce recombinant plasmid pTOPOOsHK3b, in which the hybrid histidine kinase gene - OsHK3b is isolated from its plasmid pTOPO-OSHK3b, in which said direct primer OsHk3bF identifies as 5'ATGACGTTCGCGAGGTACGC3 ', wherein said reverse primer OsHk3bF is identified as 5'CTATTCAACTTGGTCATGATTTTG3', wherein said PCR comprises a hybridization step on PCR carried out at approximately 55 ° C, wherein said OsHK3b gene has 2598 bp cDNA, wherein said OsHK3b gene is as provided in Figure 2A, wherein said chain reaction of Polymerase (PCR) comprises the steps of: i) preparation of the cDNA reaction mixture and the direct primer OsHk3bF identified as 5'ATGACGTTCGCGAGGTACGC3 'and the reverse primer OsHk3bR identified as 5'CTATTCAACTTGGTCATGATTTTG3'； ii) initial denaturation of the reaction mixture of step -i) at about 94 ° C, preferably for about 5 minutes； iii) denaturation of the reaction mixture of step-ii) at about 94 ° C, preferably for about 1 minute； iv) hybridization of the reaction mixture of step-iii) at about 55 ° C, preferably for about 1 minute； v) extension of the reaction mixture from step-iv) to about 72 ° C, preferably for about 3 minutes； and vi) final extension of the reaction mixture of step-v) at about 72 ° C, preferably for about 7 minutes, in which the denaturation, hybridization and extension stages are repeated for about 34 cycles.Un procedimiento para el aislamiento del gen de histidina quinasa de tipo híbrido - OsHK3b a partir de arroz índica IR64 p
