| China is a big country in tobacco production, with both tobacco and cigarette output ranking the first in the world. Tobacco industry plays an important role in national economy. As an industrial crops with high profit and tax revenue, tobacco occupies a high position in our national economy. Plant diseases and each kind of adverse circumstance are main factors leading to reduction in tobacco output and quality. Enhancing the quality of tobacco and improving its disease and insect resistance, and stresses tolerance are the key to address the problems of tobacco production. As the rapid development of functional genomics and biotechnology, cloning and identification of a variety of resistance, quality genes and then using them to breed many new varieties of high quality and resistance rapidly and efficiently by genetic engineering technology have become an important technology to resolve the issues of agriculture in future. However, to explore the functional genes from tobacco relating to resistance, quality and regulation, and in order to lay the foundation for tobacco molecular breeding, full length cDNA library of CB-1 including different tissues and tissues treated with different stresses induction were constructed by using SMART ( switching mechanism at 5′end of RNA transcript ) technology, which will be used to isotate resistance and quality genes for their functional studies. The results are as follows.For the purpose to separate specific functional genes of tobacco organ development and important functional genes relating to the quality formation, we collected respectively materials including organization-wide of tobacco seedlings, roots, stems, leaves, inflorescence and different developmental periods of fruit. High-quality total RNA of different tissues were extracted. Then the equivalent RNA was mixed to isolate mRNA, and full length cDNA library of different tissues was constructed by SMART. The primary library contained 2×106 cfu/ml and the recombination rate was 100%. PCR results showed that the inserts varied from 800 to 2000bp. The full-length cDNA library was stocked after amplification, and the titer of the cDNA library was estimated as 1.0×1012 cfu/ml. Ten clones were randomly selected from the library for sequencing, which showed that eight genes were full-length genes. Ten sequences were significantly similar with those registered in the NCBI nucleotide database. Among them, two were similar with hypothetical protein sequences and the six were significantly similar with known functional protein sequences which indicated that this library could be used for screening functional genes required.To isolate a variety of disease-resistant, stress-resistant functional genes of tobacco, different environmental stress treatments were carried out,such as SA, ABA, ethylene and low temperature and drought. After induction, the leaves and/or root materials in different periods respectively were taken to extract the RNA, which were mixed equivalently to be purified, and the high quality mRNA was got. And full length cDNA library of different induction was constructed using the same method. This unamplified library included 2.4×106 cfu/ml and the titer of the amplified cDNA library was estimated as 1.1×1012 cfu/ml. Twenty three clones were randomly selected from the library for identification of the recombination by PCR, and results showed that the recombination rate was 96%, The inserts varied from 750 to 2000bp. Ten clones were randomly selected from the library for sequencing, which showed that nine genes were full-length genes. Among them, the eight belonged to tobacco genes, and the other two were similar with hypothetical gene of Medicago sativa and unknown functional gene of Solanum tuberosum, which indicated that this library could be used for screening and separating specific genes. |
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