Virus-induced Gene-silencing In Wheat Spikes And The Functional Analysis Of Seed Size-related Genes TaCYP78A3 And TaCYP78A5

Wheat(Triticum aestivum L.) is one of the major staple crops for human diet. With the increase in the global population, the shortage of foods has become more and more serious. Therefore, the isolation and functional analysis of wheat yield related genes were very important to wheat breeding programs. However, traditional techniques of researching gene function can’t be widely used in wheat due to the very low transformation efficiency. In recent years, Barley stripe mosaic virus(BSMV)-based vector for wheat have been widely used for disease-resistant or pest-resistant genes silencing in seedlings. Seed size is a major component of seed yield, an important way to improve crop yield. A lot of seed size associated gene have been identified in model plant Arabidopsis and rice. However, only few genes associated with seed size has been isolated, and little is known about the mechanisms that determine the final seed size in wheat.Therefore, we first developed the system of using BSMV for gene silencing in wheat spikes, and tested the possibility of employing BSMV-VIGS to silence genes in wheat spikes. Secondly, we attempted to clone Ta CYP78A3 and Ta CYP78A5, like Glycine max Gm CYP78A3 and Arabidopsis KLU/CYP78A5, respectively by using homology cloning strategy in wheat, and further demonstrated Ta CYP78A3 and Ta CYP78A5 function in affecting seed size by using BSMV-VIGS system to silence target gene in wheat and transgenic technology to overexpressed target gene in Arabidopsis thaliana. Moreover, we try to explore the the molecular mechanisms of Ta CYP78A3 and Ta CYP78A5 affecting the final seed size by using cytological observation and molecular detection. The main topics of this thesis are as follows:1. Three hexaploid wheat(T. aestivum L.) cultivars, two winter wheat cvs Xiaoyan 6 and Shaanyou 225, and one spring wheat cv. Ningchun 16, were used in the experiments. PDS(phytoene desaturase) was treated as a reporter gene. By carrying on leaf-rub and spike-rub inoculation with the BSMV:PDS vectors carrying a 185-bp fragment of barley PDS gene, we developed the BSMV-VIGS system in wheat spikes that inoculation onto wheat spikes from heading to flowering stage was optimal for efficient silencing in wheat spikes.2. Base on BSMV-VIGS system above, the 1Bx14 gene with known function was chosen as a target gene for gene silencing on the spikes at flowering stage, with the BSMV:PDS vectors carrying a 176-bp fragment of wheat 1Bx14 gene, and then verify its biological functions. The results showed that spike-rub inoculation with the BSMV: 1Bx14 caused both m RNA and protein level of 1Bx14 significantly reduced by 85%-97% and 64%-67% in wheat cvs Xiaoyan 6 and Shaanyou 225, respectively. Moreover, 1Bx14 silencing also caused the grain gluten quality reduction. These results are consistent with previous conclusion that 1Bx14 has a positive influence on the end use quality of wheat gluten, suggesting the usefulness of BSMV-VIGS system for the functional analysis of genes expressed in wheat grains.3. By using homology cloning strategy, we first cloned two seed size related gene in wheat, Ta CYP78A3 and Ta CYP78A5 which were orthologous to Glycine max Gm CYP78A3 and Arabidopsis KLU/CYP78A5, respectively. Ta CYP78A3 and Ta CYP78A5 contained one intron and two exons, and encoded wheat cytochrome P450 CYP78A3 and CYP78A5 respectively. Their proteins both have three functional domains of the hydrophobic region, putative oxygen and heme-binding domains, which are identical to those of other CYP78 A family members.4. High expression of Ta CYP78A3 and Ta CYP78A5 were detected in young panicles immature seeds by real time PCR analysising their expression patterns. Using BSMV-VIGS system above, we silenced Ta CYP78A3 and Ta CYP78A5 expression during seed development, which caused wheat seed size reduced by 11% and 9% respectively. Further cytological observation showed that the reduction of final seed size is most likely attributable to 11% and 10% decrease in cell number of seed coat.5. In contrast, Ta CYP78A3 and Ta CYP78A5 overexpression lead to 11%-48% and 11%-35% increased seed size in Arabidopsis, respectively. Further cytological observation confirmed that the increased seed size was largely attributed to Ta CYP78A3 and Ta CYP78A5 promoting integument cell proliferation during ovule and seed development, resulting in increased cell numbers; and their expression levels had a positive correlation with the extent of integument cell proliferation. Similarly, Ta CYP78A3 and Ta CYP78A5 overexpression affected embryo development by promoting embryo integument cell proliferation during seed development, which also ultimately affected the final seed size in Arabidopsis. Unfortunately, Ta CYP78A3 and Ta CYP78A5 overexpression caused ovule developmental defect due to Ta CYP78A3 and Ta CYP78A5 overexpression promoting over proliferation of ovule integument cell, which finally resulted in reduced seed set in Arabidopsis.6. Transgenic recovery experiments further demonstrated that wheat Ta CYP78A5 is orthologous to the Arabidopsis gene CYP78A5; Ta CYP78A3 function in influencing seed and reproductive organ development but non-redundant with CYP78A9 and CYP78A5, and Ta CYP78A3 may function independently of other maternal factors Da1, TTG2, ARF2 and AP2 that influence integument cell proliferation. Furthermore, we also observed that Ta CYP78A3 and Ta CYP78A5 overexpression might lengthen longevity(2 week more than WT) of Arabidopsis, but not seed yield. Because of Ta CYP78A3/5 silencing would cause a reduction of seed size, whereas overexpressing would cause a reduction of seed set.In brief, our study is one of the first report of successful application of BSMV-based virus-induced-gene-silencing(VIGS) for gene knockdown in wheat spikes and grains, and demonstrated the feasibility of BSMV-VIGS system for the functional analysis of genes expressed in wheat grains. Applying this technology, we studied the functional characterization of wheat Ta CYP78A3 and Ta CYP78A5 gene affecting seed size. The established BSMV-VIGS system will be very useful in future research on functional analysis of genes contributing to grain quality, yield, development and disease-resistant in wheat spikes or seeds, and provided an important reference for the functional analysis of genes expressed in other monocotyledon. Moreover, the cloning and functional studies of wheat Ta CYP78A3 and Ta CYP78A5 shed light on the cellular basis of the gene influencing seed development and ultimately seed size by affecting integument cell proliferation during ovule and seed development in wheat.