Yong Gu Cho

Development and identification of transgenic rice lines with abiotic stress tolerance by using a full-length overexpressor gene hunting system.

The latest report on the draft genome of Brassica rapa sequence has been published. To elucidate the functions of these genes and to efficiently search for agriculturally useful genes, a Full-length cDNA Over-eXpressor (FOX) gene hunting system was used. The FOX library from Chinese cabbage was introduced into rice via Agrobacterium-mediated transformation. Approximately 1,150 FOX-rice lines were generated. Genomic PCR analysis indicated that the average length of FL-cDNAs introduced into individual lines was 900~1,200 bp. Basic Local Alignment System Tool (BLAST) analysis of the FL-cDNA genes revealed that 35.5% have unknown function. Most of the randomly selected transgenic rice lines showed overexpression (92%) of these genes relative to the wild-type Gopum. Moreover, 94% of the 850 transgenic rice lines were moderately tolerant (slightly yellow) to cold and 9 lines were tolerant (seedlings were light green). Morphological evaluation of the transgenic rice lines showed minimal phenotypic alteration (12%). Approximately 25.1% and 22% of the plants were significantly ahead in the days to heading and had elevated chlorophyll content, respectively. Other agronomic traits such as filled grains, number of tiller, panicle length, and culm and plant height were relatively less variable among the transgenic lines. These results provide a resource for defining genes that are associated with tolerance in transgenic rice lines.

Research on biotic and abiotic stress related genes exploration and prediction in Brassica rapa and B. oleracea: a review.

Global population is increasing day-by-day, simultaneously, crop production need to increase proportionately. Whereas, increase crop production being restricted due to abiotic and biotic stresses. Abiotic stresses are adversely affected crop growth and development, leading to crop loss globally and thereby causing huge amount of economic loss as well. Contrary, pathogens are attacked the plants imposing biotic stress and severely hampers the yield. Therefore, it is prime need to understand the molecular mechanism and genes involved to minimize the biotic and abiotic stresses for mitigating the Brassica vegetable crop losses. The stress responsive, pathogens related genes are involved in tolerance or resistance to stress in plants that are cross-talk with different types of stress components in signal transduction pathways. The plants have their own mechanism to overcome biotic and abiotic stresses to follow the abscisic acid (ABA)-dependent and ABA-independent pathways. Several transcription factors such as WRKY, Alfin-like, MYB, NAC, DREB, CBF are integrating to various stress signals and controlling the gene expression through networking with their related cis-elements. To develop stress tolerance and/or resistant crops plants, there is need to realize both of the plant and pathogenic disease development mechanisms. Therefore, this article is focused on (i) major and devastating stresses on vegetable crops, (ii) role of genes to overcome the stresses, and (iii) differential genes expressed under biotic and abiotic stresses in Brassica oleracea and B. rapa for getting insight of the mechanisms of development of resistance lines.

SP-LL-37, human antimicrobial peptide, enhances disease resistance in transgenic rice

Human LL-37 is a multifunctional antimicrobial peptide of cathelicidin family. It has been shown in recent studies that it can serve as a host’s defense against influenza A virus. We now demonstrate in this study how signal peptide LL-37 (SP-LL-37) can be used in rice resistance against bacterial leaf blight and blast. We synthesized LL-37 peptide and subcloned in a recombinant pPZP vector with pGD1 as promoter. SP-LL-37 was introduced into rice plants by Agrobacterium mediated transformation. Stable expression of SP-LL-37 in transgenic rice plants was confirmed by RT-PCR and ELISA analyses. Subcellular localization of SP-LL-37-GFP fusion protein showed evidently in intercellular space. Our data on testing for resistance to bacterial leaf blight and blast revealed that the transgenic lines are highly resistant compared to its wildtype. Our results suggest that LL-37 can be further explored to improve wide-spectrum resistance to biotic stress in rice.

Molecular characterization of the UDP-glucose 4-epimerase (BrUGE) gene family in response to biotic and abiotic stress in Chinese cabbage (Brassica rapa)

UDP-glucose 4-epimerase (UGE; EC 5.1.3.2) is an enzyme that plays an essential role in the interconverts UDP-d-glucose (UDP-Glc) and UDP-Dgalactose (UDP-Gal). Five members of the Chinese cabbage (Brassica rapa) UGE gene family, designated BrUGE1 to BrUGE5, have been cloned and characterized. Quantitative PCR shows that the BrUGE1and BrUGE4 mRNA are most abundant among other BrUGE genes, accounting for more than 55xa0% of total BrUGE transcripts in most of the tissues examined. All genes showed organ-specific expression pattern, two of which (BrUGE1 and 4) actively responded after Pectobacterium carotovorum subsp. carotovorum infection, while four genes (BrUGE-1, -3, -4, and -5)were shown to respond considerably against salt, drought and abscisic acid treatments. To better understand the function of the UGE gene, we constructed a recombinant pART vector carrying the BrUGE1 gene under the control of the CaMV 35S promoter and nos terminator and transformed using Agrobacterium tumefaciens. We then investigated BrUGE1 overexpressing rice lines at the physiological and molecular levels under biotic and abiotic stress conditions. Bioassay of T3 progeny lines of the transgenic plants in Yoshida solution containing 120xa0mM NaCl for 2xa0weeks, confirmed that the BrUGE1 enhances salt tolerance to transgenic rice plants. Also T3 progeny lines of the transgenic plants, when exposed to infection caused by Xanthomonas oryzae pv. oryzae, showed tolerance to bacterial blight. These results showed that BrUGE1 can be used as potential genetic resource for engineering Brassica with multiple stress resistance.

Identification of an SNP variation of elite tomato ( Solanum lycopersicum L.) lines using genome resequencing analysis

The completion of the tomato genome sequence and recent advances in DNA sequencing technology allow in-depth characterization of genetic variation present in the tomato genome. Cultivated tomato lines show low molecular but high phenotypic diversity. We resequenced the genomes of four elite tomato lines with an Illumina HiSeq 2000 sequencer. We generated, on average, 84 million 100-bp paired-end reads per line. Mapped reads covered 89.0-93.4% of the mapped S. lycopersicum reference genome (782 Mb). On average, 0.42 single nucleotide polymorphisms (SNPs)/kb and 0.05 short insertions and deletions (InDels)/kb were found in the four elite lines. The highest number of SNPs was 558,526 found in tomato line 13-1084. We identified many SNPs and InDels, derived from 1012 genes, in region 7-62 Mb of chromosome 5 in the four elite lines. The same pattern of multiple SNPs was detected on chromosome 5 in lines 13-1151 and 10-3321, deriving from genes in region 0-2 Mb, and many homozygous SNPs and InDels were detected on chromosome 1 of these two lines. The same pattern of multiple SNPs and InDels derived from genes located between regions 53-60 Mb of chromosomes 4 and 11 was found in the four elite lines. The SNPs and InDels identified in this resequencing study will serve as useful genetic tools and candidate polymorphisms in the search for DNA variations associated with valuable phenotypic diversity.

Overexpression of BrSAC1 encoding a phosphoinositide phosphatase isolated from Chinese cabbage ( Brassica rapa L) improved tolerance to cold, dehydration, and salt stresses in transgenic tobacco

This study demonstrates the isolation and characterization of cDNA encoding a phosphoinositide phosphatase (PIP) from a stem cell cDNA library of Chinese cabbage ( Brassica rapa ) seedling. The full length gene ( BrSAC1 ; GenBank accession no., GU434275) contained 1999 base pairs (bp), with an open reading frame of 1785 bp, encoding a polypeptide of 594 amino acids with a predicted molecular weight of 65 kDa, including a putative N-terminal signal peptide (the signal peptide counted within the 594 residues). Other regions found within the sequence include a conserved KXKXX COPI-binding motif and a consensus Cx5R (T/S) catalytic motif. BrSAC1 protein shares 92% identity with AtSac1B, and 86% identity with AtRHD4 at the amino acid level. Gene expression analyses revealed that BrsSAC1 was constitutively expressed at high levels in the pistil, stamen and flower bud, whereas it was expressed at low levels in the leaf and stem. In addition to injury, BrSAC1 expression was also induced in response to different types of stress condition, namely cold, desiccation, salt, submergence, abscisic acid and heavy metals. Overexpression of BrSAC1 in transgenic tobacco conferred tolerance to cold, dehydration, and salt stress at the seed germination/seedling stage as reflected by the percentage of germination/green seedlings, the fresh weight of seedlings and their development pattern. Our data suggest that BrSAC1 is an important stress response determinant in plants. Keywords: Abiotic stress, Brassica rapa , phosphoinositide phosphatase, transgenic plant African Journal of Biotechnology Vol. 12(15), pp. 1782-1792

Molecular characterization of BrRZFPs genes encoding C3HC4 type RING zinc finger protein under abiotic stress from Chinese cabbage (Brassica rapa L.)

Abstract The novel BrRZFPs genes encoding C3HC4- type RING zinc finger protein were identified from FOX (full length cDNA over-expressing) library of Brassica rapa . Ten full-length cDNAs obtained from the library encode zinc-finger protein containing 346 amino acids, designated BrRZFPs. These genes were classified into four groups by phylogenic analysis showing conserved protein sequences at both termini. The tissue distribution of BrRZFPs trans- cription was examined by qRT-PCR revealing ubiquitous expression pattern. However, each gene was strongly ex- pressed in the specific tissue. Transcriptional analysis showed that those acquired 10 genes were inducible under abiotic stresses. Likewise, the transcript of BrRZFP3 was strongly induced (~12-folds) by exogenous abscisic acid, whereas the transcripts of BrRZFP1, BrRZFP2 and BrRZFP3 were (> 9-folds) induced by cold. We suggest that these BrRZFPs that function as signal or response to abiotic stress are useful for crop improvement.

Enhanced bacterial resistance in transgenic tobacco expressing a BrRZFP1 encoding a C3HC4-type RING zinc finger protein from Brassica rapa

Abstract C3HC4-type RING zinc finger proteins essential in the regulation of plant processes, including responses to abiotic stresses. We previously isolated and examined the C3HC4-type RING zinc finger protein ( BrRZFP1 ) from Brassica rapa under abiotic stresses. To elucidate the role of the BrRZFP1 transcription factor in gene regulation, we transformed tobacco plants with the BrRZFP1 gene. Plants were regenerated from 82 independently transformed callus lines of tobacco and analysed for transgene expression. Trans-gene integration and expression was confirmed by Southern and RT-PCR analyses, respectively. T2 plants displayed more tolerance to the bacterial pathogens Pectobacterium carotovorum and Ralstonia solanacearum , and the tolerance levels were correlated with BrRZFP1 expression levels. These results suggest that the transcription factor BrRZFP1 is an important determinant of stress response in plants and its overexpression in plants could increase biotic stress resistance. Keywords