Modification Of Wheat Endosperm Specific Promoter And Its Application In The High Lysine Transgenic Varieties

Lysine is an essential amino acid for vertebrate, and play an very important role in the metabolism. Wheat is one of the most important crops in the world; it is also the important way to gain the plant protein. Generally, the content of lysine in wheat grain is very low, averaging only about0.44%, and the daily needed for a healthy adult is30mg·kg-1, so it can not meet the needs of the human body, and lysine amino acid plays an important role in the metabolism. Therefore lysine become the first limited essential amino acid in the grain nutrient quality of wheat, has great influence on the nutrient quality. Conventional breeding technology in crop breeding of high lysine plays a key role, especially in maize and barley, but studies show that wheat lysine content varied smaller in different varieties and interspecific, therefore acquired through a variety of hybrids and interspecific hybridization between wheat strains of high lysine has little potential. In recent years, with the development of molecular, biology quality at the molecular level of lysine in wheat breeding becomes possible at the molecular level in wheat.Search for high active promoter is the important method to improve the lysine content in wheat, this paper aims to construct a high active artificial promoter, and link with gene Cflr, construct expression vector, transform to wheat by particle bombardment, in order to obtain high lysine transgenic wheat varieties.HMW promoter is currently known high activity of endosperm specific promoter. The Bx7over expression of subunit (Bx7OE) was recently found have an important contribution to wheat quality, its promoter have a strong activity. According to the published HMW-GS Bx7gene sequence (GenBank:DQ119142.1),design primer and amplification of HMW-GS Bx7OE gene promoter by PCR. According to the database of the PLACE and PlantCARE, analyze the functional regions, the results show that:the Bx7OE promoter sequences have typical TATA box and CAAT box, seed specific promoter elements are6E-box,4G-box,7RY motifs,6B-box core sequence element. A+T62.29%>60%for rich AT region, as a general enhancer function. There are multiple light responsive element, meristem-specific activation elements, endosperm specific expression regulatory elements, a total of49specific components. In the Wild Cat,cloned promoter is43bp longer than Yangmai158and zhongyoul643bp in-1090--1047region. By function analysis, found it has a CAAT-box, it is the enhancer responsive element.Construct artificial promoter, including HMW-GS Bx7OE gene core promoter region and actin gene in rice of first intron, and the transcription start site was improved. According to the report of storage protein gene has no introns, in the promoter region which increased an intron can improve the level of gene expression, Joshi research shows that in the PCR amplification of actin3’end to increase7base GACCGCC is beneficial to improve the efficiency of transcription.7artificial promoter, Ubiquitin, Bx7OE promoter together with the GUS reporter gene fusion constructs9transient expression vector, gene gun respectively into wheat endosperm tissue, with Ubiquitin promoter for the control, GUS staining for detection of the promoter activity, and by quantitative GUS on promoter activity further validation. The results showed that:-1315--1HMW-GS Bx7promoter region contains all the activity, On gus activity,-1315--1area and actl builds artificial promoter activity is3.3times higher than Bx7OE promoter (without modification),5.6times higher than Ubiquitin.On the basis of the transformation of the promoter,we select HMW-GS Bx7OE-1315--lregion and actin gene of the first intron to construct artificial promoter, and connect with the gene Cflr (GenBank:EU367999) which has the independent intellectual property rights of lysine-rich protein in the laboratory, construction pAHC25-Bx7OEp-actl-Cflr expression vectors, by biolistics transferred into Wheat Ningmai13receptor material, Yangmai16. Use of nested primers on plant regeneration of PCR testing, up to nowdays, obtained9transgenic wheat seedlings. Through the recycling of amplified band, construct sequencing vector, sequencing results are the same with Cflr sequences. It is proved that7seedlings are positive, the next step is detecting lysine content in To generation of transgenic seeds, in order to obtain new variety of transgenic wheat which have the high lysine.