Cloning, Expression And Functional Analysis Of Non-symbiotic Hemoglobin Gene (StHb1) From Solanum Tuberosum

Potato late blight caused by Phytophthora infestans is a worldwide disease, which can lead to necrosis and decay of different parts of the whole plant, such as tuber, stem and leaf. The quality and yield of potato are influenced seriously. At present, the best promising method is to acquire the highly resistant genes to develop resistent cultivars using genetic engineering.In this paper, we preliminarily studied on correlation between StHb1gene of potato and late blight infection by semi-quantitative RT-PCR, subcellular localization analysis etc. The assay results are as following:(1) We studied the expression of StHb1gene in different resistent cultivars and tissues of potatoes by semi-quantitative RT-PCR. The results indicated that expression of StHb1gene has tissue specificity.The highest expression level of StHb1was observed in the tuber of non-stressed potato plants, and followed by root (susceptible cultivar Helan15) or stem (resistant cultivar Longshu3), while there is almost no expression in leaves and flowers.(2) To reveal inducibility of StHb1gene, the tuber tissue of resistent and susceptible cultivars were treated by zoospores suspension of P. infestans, exogenous H2O2and SNP (NO donor). Semi-quantitative RT-PCR results showed that the infection of P. infestans inhibited the expression of StHb1gene in the tuber of both resistant and susceptible cultivars against P. infestans, and exogenous nitric oxide and hydrogen peroxide also inhibited significantly. However, the degree of inhibition of StHb1gene in resistant cultivar was lower than that in susceptible cultivar.(3) A cDNA encoding non-symbiotic hemoglobin(StHb1) was cloned from Solanum tuberosum using gene specific primers, and fused to the5’end of the green fluorescent protein gene to generate plant binary vector pBI121-StHb1-GFP. It was introduced into onion epidermal cells via Agrobacterium-mediated transformation transiently. Subsequently, subcellular localization of StHb1protein was detected by fluorescent microscopy. The results showed that most of the StHb1-GFP fusion protein was localized in the nucleus, and only amall fraction was detected in the cytoplasm.(4) The prokaryotic expression vector pETHb was constructed successfully through recombination of pET-17b and StHb1. SDS-PAGE electrophoresis revealed a27.6KD protein after induction with1.0mmol/L IPTG for3hours.(5) The plant overexpression vector pBI121-StHb1and antisense expression vector pBin438-StHb1were constructed successfully.