Screening Of Arabidopsis IKU2Interacting Proteins And Analysis Of An Autotetraploid Arabidopsis

The seed is the most important reproductive organs, which plays a pivotal role in the life continuity of its species. What is more, as a nutrient reservation organ, the seed is not only the final harvest goal of most crops, but also supplies our daily life and industries with food, feed and materials. Recently, scientists have indentified a lot of key function genes through mutants and high through put gene expression analysis, but the genetic interconnection net work of those genes is rough and obscure. Polyploidy is ubiquitous in higher plant kingdom and a lot of crops are of polyploidy origin. Polyploid plants have been high valued by scientists because they can increase the yield of seeds, fruits and biomass for food, feed and biofeul production. But mechanisms of phenotypic, cellular, physiological and gene expression alterations in autopolyploid were still largely unkown. In this research, one of our studies is to characterize the interacters of a conformed seed size controlling gene HAIKU2(IKU2) by using yeast two hybrid screening, and to analyze the functions of IKU2and its interactive genes by mutants as well as transgenic plants. The other is to analyze an autotetraploid Arabidopsis isolated by our own laborotary which provided some inportant informations for autopolyploid research. The main results are as follows:1We have constructed a full length cDNA library of0-3d siliques of Arabidopsis plants. IKU2was predicted as a Leucine-rich repeat receptor-like kinase (LRR-RLK). We have obtained1candicate interacting protein AJH1by screening the library with IKU2extracellular domain (IKU2BW). What is more, we have also got27candicate interacting proteins by screening the library with IKU2intracellular domain (IKU2BN). The interactions between selected proteins and its own bait (IKUBW or IKUBN) were confirmed by colony-lift filter assay, re-mating and co-transformation. But none of those candidate protein could interact with IKU2full length protein.2We have obtained29mutants of19genes from ABRC,.Among them,18mutants related to13genes were selected with homozygotes, but none of them varies from wild type plant in seed size morphology. Transgenic analysis was mainly focused on IKU2and ORG4genes. A bunch of constructions including IKU2-OE, IKU2antisense, IKU2-GFP, IKU2BN-GFP, IKU2BW-GFP,ORG4-OE,ORG4antisense, YFP-ORG4,ORG4-GFP, ORG4-RNAi et al. were transferred with wild type Arabidopsis. Semi-quantitive RT-PCR demonstrated that most of those constructions were functional, but all of those transgenic plants did not exhibite an altered seed size and all of those proteins fused with GFP/YFP showed a negative fluorescent signal except IKU2BN-GFP.3A transgenic line IKU2antisense-49exhibited an enlarged seedling and seed size which was named as Big Seedling (BS). BS was indentified as an autotetraploid Arabidopsis.4Autotetraploid Arabidopsis BS can cause damatic phenotypic changes in organ size (dramatic increase in leaves, main stem, flowers, siliques and seeds et al.) and growth retardation from diploid WT plants. Scanning Electron Microscopy (SEM) analysis of mature seeds and Differential Interference Contrast (DIC) observations of4-11days after sowing (DAS) cotyledons revealed enlarged cotyledon size in BS plants were caused by an increase of cell expansion not by cell proliferation.5Embryo development after fertilization was repressed in autotetraploid BS by1d, but embyros at the same developmental stage were larger in BS plants than that in WT plants. Accumulations of soluable protein, fatty acid, soluable sugar and starch were increased by64%,45%,57%and55%in a single seed of BS, repectively; but that were similar to WT plants when expressed as mass fraction. Quantitive Real-time PCR (qRT-PCR) analysis of seed size related and cell cycle gene assay revealed a stable but subtle increased expression of ICK1, ICK2and ICK5by1.5-2folds. However, expressions of other genes were not statistically different between BS and WT.6BS and WT seedlings were treated with phytohormones and other substance such as glucose, mannitol and NaCl. Autotetraploid were indistinguishable from diploid plants when applied with phytohormones, but were much more sensitive to glucose treatment. However, autotetraploid were more tolerant to glucose than diploid in the germination experiments. The sensitivity in BS plants to glucose were not caused by osmotic pressure, because BS seedlings were more tolerant to NaCl, but were similar to diploid when applied with mannitol. This kind of inhibition in autotetraploid Arabidopsis was a common phenomenon because the same results were obtained in another autotetraploid plant4COL under the glucose treatments.7Compared with0.5×MS, cytological observations of the third rosette leaves and mature roots in BS and4COL plants applied with glucose exhibited an apparent suppression of cell expansion and cell proliferation simultaniously, while that of diploid plants were less affected.8Flow cytometry analysis of true leaves of BS,4COL and WT at10DAT seedlings revealed a decrease in ploidy level when applied with glucose, but a increase when applied with mannitol. Both autotetraploid and diploid plants showed similar tendency.9Compared with0.5×MS, expression levels of CYCD, CYCB, ICK1and ICK5in both autotetraploid and diploid under glucose condition were up-regulated in qRT-PCR assay, but only CYCB1.2exhibited a significant difference between autotetraploid and diploid plants. Gene expression alterations under mannitol treatment were much milder, and no different were detected between autotetrsploid and diploid.