Genetic Analysis of a Small Grain Mutant Induced by T-DNA Insertion in Rice

[Objective] The aim of this study is to understand the genetic characteristics of a grain shape mutant and its possible role in genetic improvement of grain yield in rice. [Method] On the basis of the collection of T-DNA tag lines, the progeny of homozygous plants carrying T-DNA insertion were screened for mutants with mutated phenotypes. The genetic analysis of the mutant and test for the linkage between the mutated phenotype and the T-DNA insertion were carried out to determine its genetic characteristics. [Result] In the present study, a grain shape mutant induced by T-DNA insertion in rice was identified, which showed small grain. Genetic analysis of the mutant showed that the two types of phenotype, normal and small grain in the segregating populations derived from the T-DNA heterozygotes, fit the ratio of 3∶1. Test for Basta resistance showed that all the mutants were resistant while the normal plants segregated for resistant and susceptible by the ratio of 2∶1. The results indicated that the mutant phenotype cosegregated with Bar gene. The small grain mutant caused by T-DNA insertion was confirmed by PCR amplification aiming at T-DNA. [Conclusion] The grain shape mutant is useful for isolation of the tagged gene and genetic improvement in rice.

Isolation of T-DNA flanking plant DNA from T-DNAinsertional embryo-lethal mutants of Arabidopsis thaliana by plasmid rescue technique

Three T-DNA insertional embryonic lethal mutants from NASC (The Nottingham Arabidopsis Stock Center)were first checked with their segregation ratio of abortive and normal seeds and the copy number of T-DNA insertion. The N4081 mutant has a segregation ratio of 1:3.04in average and one T-DNA insertion site according to our assay It was therefore chosen for further analysis. To isolate the joint fragment of T-DNA and plan DNA, the plasmid rescue technique waJs used. pEL-7, one of plasmids from left border of T-DNA, which contained pBR322 was selected from ampicillin plate. The T-DNA fragment of pEL-7 was checked by restriction enzyme analysis and Southern Blot. Restriction analysis confirmed the presence of known sites of EcoRI, PstI and PvuII on it.For confirming the presence of flanking plant DNA in this plasmid, pEL-7 DNA was labeled and hybridized with wild type and mutant plant DNA. The Southern Blot indicated the hybridization band in both of them. Furthermore, the junction of T-DNA/plant DNA was subcloned into bluescript SK+ and sequenced by Applied Biosystem 373A Sequencer. The results showed the 822 bp fragment contained a 274 bp sequence, which is 99.6%homolog (273bp/274 bp) to Ti plasmid pTi 15955 DNA.Ten bp of left 25 bp border repeat were also found in the juction of T-DNA and Plant DNA.Taken together, pEL-7 should contain a joint fragment of T-DNA and flanking plant DNA. This plasmid DNA could be used for the isolation of plant gene, which will be helpful to elucidate the relationship between gene function and plant embryo development.

稻瘟菌T-DNA插入所致Pi9致病突变体的获得

利用自建稻瘟菌菌株FJ95054B T-DNA插入突变体库,筛选获得2个对水稻品种P i9致病的突变体P i9-2-8T940017401和P i9-1-2 T940032801.以T-DNA侧翼序列为探针筛选该菌株的BAC文库,得到阳性BAC克隆.

Isolation of Gene Mutation from a Pathogenicity-Enhanced Mutant of Magnaporthe oryzae

To find new genes involved in fungal pathogenicity, a mutant （B11 ） exhibiting enhanced pathogenicity was isolated from an Agrobacterium-mediated transformed Magnaporthe oryzae mutant library. Southern blotting analysis showed that T-DNA insertion in the B11 genome was a single copy. TAIL-PCR and sequence alignment analyses revealed that a putative gene locus MG01679 was interrupted by the T-DNA fragment. By using the PCR-based method, the DNA and cDNA of the mutant gene MG01679 was cloned and sequenced. The open reading frame of MG01679 includes one intron and two exons, and the coding sequence is 696 bp in length and encodes a 231 amino acid peptide. Protein similarity analysis indicated that the gene belongs to the ThiJ/Pfp I protein family, and the gene was thus designated MgThiJ1. MgThiJ1 showed 57% similarity to FOXG_09029 from Fusarium oxysporum and 54% similarity to FGSG_08979 from F. graminearum in protein sequence. MgThiJ1 gene might act as a negative regulator in vegetative growth and pathogenesis in filamentous fungi, and its specific mechanism needs to be studied further.

Effort and Contribution of T-DNA Insertion Mutant Library for Rice Functional Genomics Research in China:Review and Perspective

With the completion of the rice （Oryza sativa L.） genome-sequencing project, the rice research community proposed to characterize the func- tion of every predicted gene in rice by 2020. One of the most effective and high-throughput strategies for studying gene function is to employ genetic mutations induced by insertion elements such as T-DNA or transposons. Since 1999, with support from the Ministry of Science and Technology of China for Rice Functional Genomics Programs, large-scale T-DNA insertion mutant populations have been generated in Huazhong Agricultural University, the Chinese Academy of Sciences and the Chinese Academy of Agricultural Sciences. Currently, a total of 372,346 mutant lines have been generated, and 58,226 T-DNA or Tos17 flanking sequence tags have been isolated. Using these mutant resources, more than 40 genes with potential applications in rice breeding have already been identified. These include genes involved in biotic or abiotic stress responses, nutrient metabolism, pollen development, and plant architecture. The functional analysis of these genes will not only deepen our understanding of the fundamental biological questions in rice, but will also offer valuable gene resources for developing Green Super Rice that is high-yielding with few inputs even under the poor growth conditions of many regions of Africa and Asia.

Epigenetic Suppression of T-DNA Insertion Mutants in Arabidopsis

T-DNA insertion mutants have been widely used to define gene functions in Arabidopsis and in other plants. Here, we report an unexpected phenomenon of epigenetic suppression of T-DNA insertion mutants in Arabidopsis. When the two T-DNA insertion mutants, yucl-1 and ag-TD, were crossed together, the defects in all of the ag-TD plants in the F2 popumation were partially suppressed regardless of the presence of yucl-1. Conversion of ag-TD to the suppressed ag- TD （named as ag-TD＊） did not follow the laws of Mendelian genetics. The ag-TD＊ could be stably transmitted for many generations without reverting to ag-TD, and ag-TD^＊ had the capacity to convert ag-TD to ag-TD^＊. We show that epige-netic suppression of T-DNA mutants is not a rare event, but certain structural features in the T-DNA mutants are needed in order for the suppression to take place. The suppressed T-DNA mutants we observed were all intronic T-DNA mutants and the T-DNA fragments in both the trigger T-DNA as well as in the suppressed T-DNA shared stretches of identical sequences. We demonstrate that the suppression of intronic T-DNA mutants is mediated by trans-interactions between two T-DNA insertions. This work shows that caution is needed when intronic T-DNA mutants are used.

A gene encoding AtPIP5K2 may be involved in regulating the sensitivity to osmotic stress

One mutant line eto with salt tolerance was screened from a T-DNA insertion mutant collection of Arabidopsis thaliana. In addition to a reduced rate of seed germination, NaCl and ABA also inhibited the growth and the greening of cotyledons of wild-type seedlings, but not the eto mutant. TAIL-PCR analysis showed that T-DNA tag insertion in the eto was located at nucleotide 27,502 in BAC F3M18, upstream （at position -487 relative to the translation initiation codon） of gene At lg77740 （encoding a putative phosphatidylinositol-4-phosphate 5-kinase, AtPIP5K2）. This inserted mutation cosegregated closely with the eto phenotype, Another analysis not only indicated that AtPIP5K2 transcript is expressed predominantly in roots and rosette leaves, but also showed the T-DNA insertion resulted higher accumulation of the AtPIP5K2 in eto mutant plants and did not influenced the expression of the upstream At lg77730 gene. This change may play an essential role in the tolerance of eto mutant plant to the osmotic stress.

Molecular analysis of rice plants harboring a multi-functional T-DNA tagging system

About 25,000 rice T-DNA insertional mutant lines were generated using the vector pCAS04 which has both promoter-trapping and activation-tagging function. Southern blot analysis revealed that about 40% of these mutants were single copy integration and the average T-DNA insertion number was 2.28. By extensive phenotyping in the field, quite a number of agronomically important mutants were obtained. Histochemical GUS assay with 4,310 primary mutants revealed that the GUS-staining frequency was higher than that of the previous reports in various tissues and especially high in flowers. The T-DNA flanking sequences of some mutants were isolated and the T-DNA insertion sites were mapped to the rice genome. The flanking sequence analysis demonstrated the different integration pattern of the right border and left border into rice genome. Compared with Arabidopsis and poplar, it is much varied in the T-DNA border junctions in rice.

Phenotypical and structural characterization of the Arabidopsis mutant involved in shoot apical meristem

An Arabidopsis mutant induced by T-DNA insertion was studied with respect to its phenotype,microstructure of shoot apical meristem(SAM)and histochemical localization of the GUS gene in comparison with the wild type.Phenotypical observation found that the mutant exhibited a dwarf phenotype with smaller organs(such as smaller leaves,shorter petioles),and slower development and flowering time compared to the wild type.Optical microscopic analysis of the mutant showed that it had a smaller and more flattened SAM,with reduced cell layers and a shortened distance between two leaf primordia compared with the wild type.In addition,analysis of the histo-chemical localization of the GUS gene revealed that it was specifically expressed in the SAM and the vascular tissue of the mutant,which suggests that the gene trapped by T-DNA may function in the SAM,and T-DNA insertion could influence the functional activity of the related gene in the mutant,leading to alterations in the SAM and a series of phenotypes in the mutant.