Investigation Of Lipoxygenase Activity And Cloning Of Lipoxygenase Genes And Their Function Analysis In Chinese Bread Wheat (Triticum Aestivum L.)

Flour color is one of the most important wheat quality traits and has an important effect on qualities of noodles, steamed bread and other related products. Lipoxygenase(Lox) in grain is an important factor affecting flour color and the processing quality of wheat-based products, and has now become one of the important goals in wheat quality breeding. Study of Lox activity will be beneficial for the improvement of wheat products, flour processing quality, extending the storage time of seeds and so on. Dissection of molecular and genetic mechanism of Lox activity is of the great significance on wheat quality improvement in China.In the present study, a F4:5 recombinant inbred lines(RIL) population encompassing 110 lines derived from Xianyang 83104 / Zhoumai 16 and 163 winter wheat cultivars and advanced lines from the Yellow and Huai valleys were used to identifiy Lox activity and clone Lox genes by in-silico cloning, spectrophotometer, quantitative real-time PCR, RNA interference, gene genetic transformation, PCR amplification with specific primers, site-specific cleavage with the restriction enzyme, molecular cloning, DNA sequencing, etc. Based on the Lox genes sequence differences, we developed a functional marker. The main results obtained in this study are summarized below.1. Winter wheat cultivars or lines from the Yellow and Huai valleys mainly have medium Lox activity. Significant difference in grain Lox activity was observed in different cultivars or lines. Grain Lox activity among cultivars or lines ranges from 49.13 to 110.77 A234 min-1g-1, and has a large selection potential. Analysis of variance on grain Lox activity in 163 winter wheat cultivars with different genetic background from the Yellow and Huai valleys in different environment conditions showed that there is significant difference amongst grain Lox activities of different years and locations. The grain Lox activity of different cultivars or lines across six environments has a high heritability(h2=0.75), indicating that the effects of genotype, environment and their interactions on Lox activity are all significant and that the genotype is the predominant factor to determine Lox activity of wheat grain.Lox activity of wheat grain not only influences flour color, but also has a significant effect on other quality traits. Grain Lox activity was significantly negatively correlated to flour lightness, and was significantly positively correlated to flour yellowness, but has no significant correlation with protein content, grain hardness, wet gluten content and other quality traits. These studies could provide useful information for further understanding the correlations between grain Lox activity and flour color as well as other quality traits.2. Ta Lox-B2 and Ta Lox-B3 genes on the chromosome 4BS in bread wheat were cloned by in-silico cloning technology. The full length of genomic DNA sequence of Ta Lox-B2 possesses 4267 bp, and Ta Lox-B3 gene genomic DNA sequences possesses 4246 bp, and their similarity at the DNA level is 84.8%. Both Ta Lox-B2 and Ta Lox-B3 genes comprise 6 introns and 7 exons. Their full-length c DNA are 2586 bp. The deduced amino acid sequence showed that the similarity of Ta Lox-B2 and Ta Lox-B3 genes is as much as 97.8%, and both Ta Lox-B2 and Ta Lox-B3 genes encode an 861 amino acid protein possessing a lipoxygenase superfamily domain at the 170-838 interval.3. Two different Ta Lox-B2 alleles, designated Ta Lox-B2 a and Ta Lox-B2 b, were subsequently discovered. Based on the sequences of Ta Lox-B2 a, Ta Lox-B2 b and Ta Lox-B2 genes, a co-dominant marker, Lox-B23, was developed to precisely distinguish these three alleles in Chinese bread cultivars. Both 788-bp and 677-bp fragments were amplified from the cultivars with Ta Lox-B2 a and Ta Lox-B3 showing higher Lox activity, respectively. Cultivars with Ta Lox-B2 b allele(Amplified 660-bp fragment) s show lower Lox activity. We found a total of five allelic combinations of Lox genes at Ta Lox-B1, Ta Lox-B2, and Ta Lox-B3 loci on chromosome 4BS in wheat cultivars surveyed. Bread wheat cultivars with Ta Lox-B1a/Ta Lox-B2a/Ta Lox-B3 a combination exhibited the highest Lox activity(75.41 A234 min-1g-1), whereas cultivars with Ta Lox-B1a/Ta Lox-B2b/Ta Lox-B3 b combination showed the significantly lowest Lox activity(72.25 A234 min-1g-1). The marker Lox-B23 was mapped on chromosome 4BS using a set of Chinese Spring nullisomic-tetrasomic lines, Chinese Spring ditelosomic lines(CS Dt 4AS/4AL, CS Dt 4BS/4BL, CS Dt 4DS/4DL). The relationship between Ta Lox-B3 gene and grains Lox activity was furtherly comfirmed using a RIL population derived from Xianyang 83104 / Zhoumai 16, and the results showed that Ta Lox-B3 gene significantly increased grain Lox activity.4. Quantitative real-time PCR was conducted to research m RNA relative expressing levels of Ta Lox-B2 and Ta Lox-B3 genes in different organs of wheat, in different stress conditions and grain development of different stage. The results showed that Ta Lox-B2(Ta Lox-B2 a or Ta Lox-B2b) and Ta Lox-B3(Ta Lox-B3a) genes could express in various organs of wheat. Ta Lox-B2 gene expression was mainly in the leaves, while Ta Lox-B3 was expressed primarily in the roots. Expression trend of Ta Lox-B2 a and Ta Lox-B3 genes were significantly different in 4℃ low temperature, 42℃ high temperature, Na Cl and PEG 6000 stress conditions. The Ta Lox-B2 a gene expression decreased at 42 °C treatment from 0 h to 6 h with symmetrical trendline under Na Cl treatment similar to that of Ta Lox-B3 a gene. However, the Ta Lox-B2 a and Ta Lox-B3 a genes did not show a significant trendline under PEG 6000 treatment, and their expression levels were very low. Quantitative real-time PCR revealed that Ta Lox-B2 a and Ta Lox-B3 a genes could express in various organs of wheat, and their expression levels were affected by temperature and Na Cl stress. Additionally, expression profiles of the Ta Lox-B2 and Ta Lox-B3 genes in seeds of the different developmental stages were shown in a Chinese current elite cultivar Aikang 58 with Ta Lox-B2 a and Ta Lox-B3 a genes. Relative expression levels of the two genes were the highest in the 7th day after anthesis of wheat grain. However, Ta Lox-B2 a and Ta Lox-B3 a genes expression profiles were different trends during wheat grain development of different stages.5. RNAi expression vector of Ta Lox-B1 gene was constructed successfully to conduct genetic transformation in bread wheat in order to comfirm the influence of Ta Lox-B1 gene on Lox activity in bread wheat. Totals of 1690 immature embryo callus were bombarded by gene gun, and final evaluation obtained 21 generation transgenic plants, and the average conversion rate was very low, only, i.e. 1.2%. Lox activity of generation transgenic T3 seeds significantly reduced compared to control plants. It suggests that the missing of expression product of Ta Lox-B1 gene led to the reducing of Lox activity in generation transgenic wheat grain. Furthermore, the size of the transgenic wheat seedlingsand the numbers of leaf and tiller were less than the control plants(wild type). RNAi technology not only leaded to the decrease of wheat grain Lox activity significantly, but also changed their agronomic traits. Quantitative RT-PCR results showed that the expression of Ta Lox-B1 gene of transgenic wheat plants was significantly lower than that of control plants. This indicates that the insert of introduced gene has efficiently restrained the expression of lipoxygenase gene.