Extraction And Purification Of β-glucan, RAPD Analysis Of The Varieties And Cloning And Expression Of HBGHⅡ Gene From Tibetan Hulless Barley

As one of the origins, China has a large quantities of barley, especially the hulless barley, which shows an important status in the world. Hulless barley is mainly distributed in Qingzang highland,including Tibet;Qinghai;Ganshu, Yunnan and north-west Sichuan. Recently, it received much attention for its seeds' P -glucan content, for high P -glucan content varieties can be used to make new medicine and functional food. The β-1, 3-glucanase (1,3-β-D-glucan 3-glucano-hydrolase; EC 3.2.1.39) family of proteins has received much attention in these areas of classification, physiology, anti fungi, molecular biology and genetic engineering.The P -glucan contents of 34 varieties of Tibetan hulless barley from Tibet,Gansu, Yunnan and Sichuan were detected by the enzyme method. The β -glucan total contents varied from 3.87%-l 1.96%.Among them, 23 varieties' β -glucan total content exceeded 5%,about 67.6% of the whole varieties. And the content of soluble β -glucan exceeded the insoluble one (except several varieties).we use hot water and enzyme method to isolate β -glucan from Tibetan hulless barley. The biochemical properties of the polysaccharide product was analysed. The results of paper chromatography, UV absorbance and IR chromatography showed that the isolated polysaccharide is relatively pure, and the image was detected by SEM as well. It was considered that the extracting method was ideal for isolating β -glucan from Tibetan hulless barley for its simplicity and high efficiency.RAPD (random amplified polymorphic DNA) markers were generated from 23 Tibet hulless barley varieties including 21 breeding varieties and 2 farmyard varieties. Among the total 48 fragments generated by 5 selected primers (among 68 primers), 44 appeared to be polymorphic (92%). Cluster analysis was performed by software RAPDistance 1.04. The 23 varieties were divided into 2 groups. The molecular foundation of genetic diversity was explored. In addition, one DNA fingerprinting based on 17 bands amplified with S32 and 2 bands with S18 was constructed for identification all the varieties.A new full-length Tibet hulless barley β-1, 3-glucanase cDNA (GenBank accession No. AF515785) was obtained by RT-PCR and RACE techniques, and its complete gene sequence obtained by DNA Walking (GenBank accession No. AY178838). Sequence alignment with the BLAST program shows that cDNA has highly similarity (99% identity) with barley β-1,3-ghicanase GIl. It encodes a major open reading frame of 334 amino acids, preprotein of 35kDa; the mature protein consists of 306aa with peptide of the first 28aa,; 5' untranslated regions contains 46nt, 3' untranslated regions contains 165nt, a Poly (A) tail signal and long Poly (A) tail. DNA sequence indicated it has 2 produced TATA boxes,4 CAAT boxes and 4 GC boxes,and an intron of 165bp between signal peptide and mature protein. Southern blot analysis indicated that the gene is a member of a small gene family. RT-PCR and northern blot analysis indicated it is constirutively expressed in barley shoots. Thus it is a new member of β-1, 3-glucanase gene family. The information we have got is very important for the further research of gene structure, expression and fundation of β-1, 3-glucanase II.For Prokaryotic expression, the mature portion and total protein of the β-1, 3-glucanase II was engineered by PCR. The gene was transferred to an E.coli strain Ml5 vector expression behind the T5 promoter -QIAexpress System pQE-30. The recombinant gave rise to a 33KD mature protein in response to the IPTG induction; its content was about 12.4% among total cell protein by Gene Genius Bio Imaging System. The induced protein was purified by Ni-NTA matrices and subjected to SDS-PAGE and the recombinant protein catalyses the hydrolysis of β-1, 3-glucan with an action pattern characteristic of a β-1, 3-glucan endohydrolase (EC3.2.1.39) with 1800 nkat (nanokatals) per mg protein activity.The anti-fungal activity of purified β-1, 3-glucanase was detected by Gibberelle Zeae(Schw.) Fetch, Curvularia Lunat...