The Cloning And Function Analysis Of A CMS Specific Hsf CDNA In Capsicum Annuum9704A

The cytoplasm male sterility (CMS) is a widespread natural phenomenon in plants. It provides the good female parents for crop heterosis utilization by reduce the costs of artificial emasculation and blocking its selfing contaminant in hybridization. The pepper cytoplasmic male sterile9704A is a male sterility stable line breeded by Hunan Institute of Vegetable Science. It has a good application as maternal material in chili pepper cross breeding.A heat shock transcription factor (Hsf) of pepper9704A was identified by Solexa transcriptome sequencing in expression differences compare to its homogenous maintainer line9704B previously. The gene cDNA was cloned by RT-PCR combined with RACE and be sequenced. The sequence showed that the cloned Hsf cDNA is in2018bp with the coding region1083bp and codes a360amino acid putative protein character as a heat shock transcription factor. Gene homology analysis showed that the gene is one of the plants Hsf gene family members and shares90%homology with the Hsf in tomato and73%identity in amino acids with Hsf6AB in Arabidopsis thaliana. The real-time fluorescence quantitative RT-PCR of the gene expression was carried out with the compared tissues in9704A and9704B. The Hsf gene expressed very low in the stems of the two materials and higher in both of the roots. The obvious difference was that the gene expression in9704A leaf is13times higher than it is in the maintainer line and the flower bud expression is in3times higher. According to the function of Hsf that we speculated the Hsf gene in high level expression in sterile leaf may correlate the CMS by regulating the chloroplast or mitochondria targeting heat shock proteins (Hsp) and affect the normal function of chloroplasts and mitochondria.In order to further study the function of the gene we constructed the Hsf gene overexpression vector in pWM101as pWM10-Hsf and the antisense expression inhibition vector pWM101-anti-Hsf The two recombinants were transformed into tobacco WS38respectively by leaf disco-culturation. Some transgenic tobacco plantlets of the two constructions are screened out and identified by molecular test.The regeneration rates, root inducing rates of the overexpression Hsf transgenics are apparently lower than the antisense transgenics. The plant height and growth potential is weeker than that of the antisense transgenics and wild control. While the antisense transgenics are very like the wild counterparts.The pollen fertilization ratios are tested by potassium iodide staining method when transgenic plantlets are in flowering. The statistics show that pollen amounts are significantly less in the mature anther of the overexpression Hsf transgenics than it is in the antisense transgenics and wild WS38. The stained pollen is only23.44%in the overexpression transgenic tobacco while it is77.03%in the antisense transgenic and80.03%in the wild. It further verified that the excessive expression of Hsf may lead to sterility of the pepper. We harvested the seeds of the transgenic tobacco and treated the early seedlings in different temperature in a3h duration. The overexpression transgenic seedlings survivl ratio is96.5%in50℃while all the seedlings of antisense transgenics and the wild lethal. It demonstrated the early seedling of the overexpression transgenics are highly heat resistance due to the Hsf overexpression.We concluded that the Hsf increase the heat resistance of the plant but simultaneously reduce its fertilization. It is a phenomenon of the heat adaption of the pepper in its evoluation.