| Flavonoids are the main components for the medical effect in Sausurrea medusa. The key Enzymes for the synthesis pathway of plant flavonoids include phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), and a number of regulatory genes as some Myb family genes mediate the transcriptional activation of the flavonoids biosynthesis. We hope to gain a higher production of flavonoids through transforming these genes into suspension cells of Sausurrea medusa, and provide a solid ground on technology for further large scale produce of suspension cells.Sausurrea medusa, which live in the high and inclement environments, have an excellent resistance character against many adversities. The genes that related to these resistance characters in Sausurrea medusa would be of great economic value on crop cultivation. We can improve the adversities resistance of resource plants and crops by transforming these genes into them.Several complete or partial genes cDNA were cloned from cDNA library of Sausurrea medusa, and two new methods for screening cDNA library were established in this paper. All the results as the following:1. Subsection method for cDNA library screening. With this method, cDNA phage plate was cut into several blocks, dilute each section using dilution buffer and detect them by PCR respectively. Then the target genes were obtained by screening cDNA library. Comparing with other methods, this method has many advantages such as controlled range and clear target, and also quick and simple for obtaining the target genes. It is possible to get a target gene in one week in general by this method. We can get twice the result with half the effort when we screen several genes in the same time. It is also suitable to screen other libraries.2. Another method-creening several genes from cDNA library using one pair of primers by PCR was brought forward here. In this method, a pair of primers have been designed basing on the same or the similar parts of several sequences, and been used to screen over two genes from cDNA library by PCR at the same time. It's a good method for cloning such genes involved some similar or same partial sequences, and could be applied to screening some other libraries.3. One chalcone isomerase gene (No: AF509335) was cloned from cDNA library. This gene involves 831bp, 57% a and t, a polyA signal, and one 31 bp polyA tail. One 699bp open reading-5 CHI jfflM)^^ - Abstractframe was found then. Putative protein includes 232 amino acids and 20% Ser and Thr, pi 4.15.4. A Flavanone 3-hydroxylase (No: AF509338) complete cDNA was isolated from cDNA library, which is 1344bp length in bases sequence, a and t are higher than c and g in bases content, a polyA signal and 31bp polyA be involved in it. Putative protein includes 343 amino acids, pi 5.55. Only 30-40% conserved parts have been found in the bases sequence comparing with other genes, which is far lower than that when comparing with each other in themselves. Some similar domains to Isopenicillin N synthase and partial similar domains to 2OG-Fe(II) oxygenase superfamily were found when Search the protein conserved domains in GenBank.. Protein Blast shows that it has 40-50% identities with dioxygenase (eggplant), hyoscyamine 6-beta-hydroxylase (Atropa belladonna), 2-oxoglutarate-dependent dioxygenase (Solanum chacoense), hyoscyamine (6S)-dioxygenase (henbane), hyoscyamine 6-beta-hydroxylase-like protein (Solanum tuberosum), flavanone 3-hydroxylase (Oryza sativa), flavanone 3-beta-hydroxylase (Arabidopsis thaliana), putative Fe(II)/ascorbate oxidase (Arabidopsis thaliana), and some other enzymes. These hint that this gene may functions as completely distinct action with such F3H we have known.5. One HSP70 gene complete cDNA (No: AF509336 ) and a partial cDNA were cloned from library. 2224bp be involved in the complete cDNA and 1484bp in the partial cDNA. Putative protein includes 647 amino acids in the complete HSP70, and its' MW is 70715 D, PI 4.95. Several charact...
|
PDF Full Text Request