ORF43 (ac43) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved baculovirus gene whose function is unknown. To determine the role of ac43 in baculovirus life cycle, we used a new AcMNPV bacmid (bAc-MK) and generated ac43 deletion virus (ac43KO) by using the plasmid capture system (PCS). After transfection into Spodoptera frugiperda cells, ac43KO produced significantly different OBs which with much larger size; and especially had much single nucleocapsids compared to Ac-MK. Furthermore, ac43KO bacmid led to defect in transcription and expression of polyhedrin, which result in less OBs production. However, ac43KO didn't affect BV production since there's no remarkable difference of BV titer in both ac43KO and Ac-MK. These results demonstrate that ac43 play an important role in polyhedrin expression, OB formation, and virion assembly.

In the previous study, we found an orange-eyed mutant of the brown planthopper (BPH). And we confirmed that it’s orange phenotype is controlled by single recessive allele in the autosome. To understand the effect of the orange eye color on mating of BPH, we designed two different combinations of the mate choice test with three virgin female and male BPH adults. The one consisted of orange-eyed female (org/org), orange-eyed male (org/org), and homozygous normal-eyed male (+/+) (female mate choice). The other was composed of orange-eyed male (org/org), orange-eyed female (org/org), and homozygous normal-eyed female (+/+) (male mate choice). In female mate choice test, four mating types could be distinguished according to the distribution of the eye-color phenotype in F1 progeny and their hatching-order in each mating pair. Two mating types showed only one eye-color phenotype, normal and orange, respectively, and the other two produced both eye-color phenotypes in a different hatching-order. In male mate choice test, both phenotypes of offsprings were also produced in most mating pairs. From these results, the effect of eye color on mating of BPH was not clearly found, but the multiple mating in both sexes and the mating preference by female are cautiously suggested.

Pleurotus ostreatus 'Miso' is a mutant strain showing white color in pileus from the known parent strain 'Wonhyeong No. 1'. Shape and several other characters also vary with culture conditions. Mating experiments were performed to understand interstrain mating relationship using monokaryons of the parent and the mutant strains. All monokaryons were grown from single spores isolated from freshly collected fruit bodies. Pairings were performed in 90 mm petri dishes on PDA. They were allowed to grow at 25℃ until two fronts of the advancing mycelia met and developed a conspicuous contact zone. The contact zone and the outer edges of paired colonies on each plate were examined for clamp connections. The crosses were considered positive when clamp connections were at the growing margin of either side of the interacting monokaryons. The parent strain 'Wonhyeong No. 1' and the mutant strain 'Miso' resulted in tetrapolar incompatibility system in intrastrain crosses. Normal compatible matings could be visibly distinguished from incompatible ones by the rapid growth and gross morphology. The resultant dikaryotic colony was also confirmed microscopically by the presence of clamp connections from incompatible pairings. In interstrain crosses, each monokaryotic tester strains of the parent strain was out-crossed to monokaryotic tester strains of the mutant. The four classes of tester strains representing the four incompatibility types were chosen from each mutant and parent strains. As a result of these crosses it was found that both strains share the same A and B incompatibility factors yielding 25% compatibility.

Autographa californica nucleopolyhedrovirus (AcMNPV) has a large doublestrand DNA genome of approximately 134 kbp and comprises 156 open reading frames (ORFs). To elucidate DNA replication cascade of AcMNPV, we developed a novel baculovirus genome that can be maintained in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. This genome, named bAc-MK, contains a mini-F replicon and a kanamycin resistance marker. Using a convenient Tn7 transposon-based system, pPCS-S, which contains an ampicillin resistance gene, 56 single ORF-truncated mutants were generated by random insertion into bAc-MK genome. These single ORF-truncated mutants were independently transfected into Sf9 cells to verify viral replication. Interestingly, both lef-1 and p48 knockout mutants showed normal viral replication in infected cells, which are reported to essential for viral replication. These results suggest that these single ORF-truncated mutants are useful for elucidation of viral replication cascade.

Mutations in DLX3 are associated with both autosomal dominant hypoplastic hypomaturation amelogenesis imperfecta (ADHHAI) and tricho-dento-osseous (TDO) syndrome. ADHHAI is caused by a c.561_562delCT (2bp-del DLX3) mutation whereas TDO syndrome is associated with a c.571_574delGGGG (4bp-del DLX3) mutation. However, although the causal relationships between DLX3 and an enamel phenotype have been established, the pathophysiological role of DLX3 mutations in enamel development has not yet been clarified. In our current study, we prepared expression vectors for wild type and deletion mutant DLX3 products (4bp-del DLX3, 2bp-del DLX3) and examined the effects of their overexpression on the expression of the enamel matrix proteins and proteases. Wild type DLX3 enhanced the expression of matrix metalloprotease 20 (MMP20) mRNA and protein in murine ameloblast-like cells. However, neither a 4bp-del nor 2bp-del DLX3 increased MMP20 expression. Wild type DLX3, but not the above DLX3 mutants, also increased the activity of reporters containing 1.5 kb or 0.5 kb of the MMP20 promoter. An examination of protein stability showed that the half-life of wild type DLX3 protein was less than 12 h whilst that of both deletion mutants was longer than 24 h. Endogenous Dlx3 was also found to be continuously expressed during ameloblast differentiation. Since inactivating mutations in the gene encoding MMP20 are associated with amelogenesis imperfecta, the inability of 4bp-del or 2bp-del DLX3 to induce MMP20 expression suggests a possible involvement of such mutations in the enamel phenotype associated with TDO syndrome or ADHHAI.

Dlx3 is a homeodomain protein and is known to play a role in development and differentiation of many tissues. Deletion of four base pairs in DLX3 (NT3198) is causally related to tricho-dento-osseous (TDO) syndrome (OMIM #190320), a genetic disorder manifested by taurodontism, hair abnormalities, and increased bone density in the cranium. The molecular mechanisms that explain the phenotypic characteristics of TDO syndrome have not been clearly determined. In this study, we examined phenotypic characteristics of wild type DLX3(wtDlx3) and 4-BP DEL DLX3 (TDO mtDlx3) in C2C12 cells. To investigate how wtDlx3 and TDO mtDlx3 differentially regulate osteoblastic differentiation, reporter assays were performed by using luciferase reporters containing the promoters of alkaline phosphatase, bone sialoprotein or osteocalcin. Both wtDlx3 and TDO mtDlx3 enhanced significantly all the reporter activities but the effect of mtDlx3 was much weaker than that of wtDlx3. In spite of these differences in reporter activity, electrophoretic mobility shift assay showed that both wtDlx3 and TDO mtDlx3 formed similar amounts of DNA binding complexes with Dlx3 binding consensus sequence or with ALP promoter oligonucleotide bearing the Dlx3 binding core sequence. TDO mtDlx3 exhibits a longer half-life than wtDlx3 and it corresponds to PESTfind analysis result showing that potential PEST sequence was missed in carboxy terminal of TDO mtDlx3. In addition, co-immunoprecipitation demonstrated that TDO mtDlx3 binds to Msx2 more strongly than wtDlx3. Taken together, though TDO mtDlx3 acted as a weaker transcriptional activator than wtDlx3 in osteoblastic cells, there is possibility that during in vivo osteoblast differentiation TDO mtDlx3 may antagonize transcriptional repressor activity of Msx2 more effectively and for longer period than wtDlx3, resulting in enhancement of osteoblast differentiation.

To investigate the antifungal activity related protein in pesticidal bacteria, a bacterial strain LTD was isolated from soil collected at Gimje in Jeonbuk province, Korea, and identified as Bacillus subtilis LTD based on a API50 CHB kit and 168 rDNA seque

Pythium ultimum에 의한 뿌리썩음병을 생물학적으로 방제하기 위하여 이에 대한 항진균 활성이 있는 세균 Bacillus lentimorbus WJ5를 방사선 (60Co)을 조사하여 보다 더 강한 항진균 활성을 가지는 돌연변이체 B. lentimorbus WJ5a17을 유도하였다. B. lentimorbus WJ5와 WJ5a17을 각각 활용한 미생물제제 FWJ5와 FWJ5a17을 조제하였으며 균체수는 1.0× 1011

xylA 유전자의 발현에 관한 xylR 유전자의 조절 메카니즘을 밝히기 위한 연구의 일환으로 xylA 프로모터 하류에 cat 유전자를 삽입시켜 Pxyl-cat-xylA 융합 플라스미드인 pEXC131을 제작하였고 이 플라스미드를 xylA 변이주인DH77로 형질전환시킨 결과 xylose의 유도시에만 Cm 내성과 xylose isomerase활성이 나타났다. pEXC1131/DH77에 NTG를 처리하여 xylose 유도없이도 Cm 내성과 xylose isomerase의 활성을 나타내는 xylA 유전자의 구성적 변이주인 pEXC131-39를 xylR 변이주인 DH60으로 형질전환시킨 균주가 xylose에 의한 유도와 무관하게 Cm 내성 및 xylose isomerase 활성을 가지는 것으로 보아 xylA 유전자의 프로모터부위의 변이에 의한 구성적 변이주임을 확인하였다.

1975년 수원 잠업시험장 상전에서 뽕나무 세균성위축병균 Psendomonas mori(Boyer et Lambert) Stevens의 형태적으로 다른 Rough colony형(R형) 균주를 분리하였다. 이를 전국 5개 지역에서 채집, 분리한 wild type인 Smooth colony형 (S형) 균주와 병원성 및 세균의 일반적 성질을 비교하고 유전적인 안전성을 검토한 결과는 다음과 같다. 1. R형균주는 S형균주에 비하여 강한 병원성을 보였으나 병징에는 아무런 차이가 없었다. 2. R형균주는 그 길이가 200u 이상에 달하는 사상하며 편모가 없는 비운동성 세균으로, 단간상이고 운동성이 있는 S형균주와 형태적으로 달랐다. 3. 육즙한천배지에서 S형균주와 colony는 백색, 원형, 전록으로 평골한데 비하여, R형균주의 colony는 주변이 불규칙한 파상으로 그 중앙부위에 많은 주름무늬가 있다. 4. 46종의 생리적 성질 및 혈청학적 성질에서는 R형과 S형균주 사이에 아무런 차이가 없었다. 5. R형과 S형균주 모두 100회 이상의 계대배양이나 의 NaCl 및 유기산의 Sodium 염 5종, 항생물질 4종에 의한 처리에서 유전적인 안정성을 보였다.

Developing rice lines with various amylose contents is necessary to diverse usages of rice in terms of raw materials for processed food production, and thereby to promote rice consumption in Korea. A rice mutant line, ‘Namil(SA)-dull1’ was established through sodium azide mutagenesis on ‘Namil’, a non-glutinous Korean Japonica rice cultivar. Namil(SA)-dull1’ had dull endosperm characteristics and the evaluated amylose content was 12.2%. A total of 94 F2 progenies from a cross between ‘Namil(SA)-dull1’ and ‘Milyang23’, a non-glutinous Tongil-type rice cultivar, was used for genetic studies on the endosperm amylose content. Association analyses, between marker genotypes of 53 SSR anchor markers and evaluated amylose contents of each 94 F2:3 seeds, initially localized rice chromosome 6 as the harboring place for the modified allele(s) directing low amylose content of ‘Namil(SA)-dull1’. By increasing SSR marker density on the putative chromosomal region followed by association analyses, the target region was narrowed down 0.94 Mbp segment, expanding from 28.95 Mbp to 29.89 Mbp, on rice chromosome 6 pseudomolecule. Among the SSR loci, RM7555 explained 84.2% of total variation of amylose contents in the F2 population. Further physical mapping on the target region directing low amylose content of ‘Namil(SA)-dull1’ would increase the breeding efficiency in developing promising rice cultivars with various endosperm characteristics.

In recent years, the efficiency and accuracy of QTL analysis for identification of useful traits have been increased by high-throughput genotyping. In a previous study, the genome variation of significant DNA polymorphism was observed in early maturing type rice mutant (EMT) by comparing with that of wild type (WT). For detection of major QTL for flowering time, we constructed a linkage map of 36 InDel- and 6 SNP- markers. In the linkage analysis of F2 plants derived from the cross “WT x EMT”, we have detected one potential QTL region on chromosome 6 by M6-3 marker. Also, the Hd1, which contained the target fragment of M6-3 marker, exhibited the relatively high nonsynonymous substitutions in genes located on chromosomal region from M6-2 to M6-4. To evaluate the reliable allele segregation related to expected Mendelian ratio between M6-3 and its flanking markers, M6-3 marker developed in Hd1 gene exhibited the 1:2:1 ratio as clear monogenic segregation in heterozygous F3 plant. Additionally, we further analyzed the different transcript regulations of OsGI and Hd3a gene related to Hd1 involved in photoperiodic flowering pathway. Although the mRNA levels of Hd1 had no difference between WT and EMT, the Hd3a as downstream effector of Hd1 significantly upregulated in EMT, suggesting that Hd1 gene may become nonfunctional.

Brassinosteroids (BRs) control virtually every aspect of plant growth and development. BRs act alone or with other exogenous and endogenous signals including auxin and light. To screen for the novel player involved in BR signaling in Arabidopsis, we employed cDNA overexpression strategy. We created a cDNA library to be expressed under the 35S overexpression promoter, and introduced into a weak brassinosteroid insensitive 1 (bri1) mutant. The mutant dubbed bri1-5 with long petiole (blp) was identified to display bigger stature especially in hypocotyl and petiole length relative to bri1-5. Sequence analysis of the rescued transgene revealed that blp consisted of a chimeric DNA consisting of a 3’ half of PHYB, 2 bp insertion, and a part of a chloroplast ribosomal RNA. Re-introduction of chimeric DNA into bri1-5 recapitulated blp phenotype. The blp phenotypes being similar to phyB mutants led us to examine both the PHYB transcript and protein levels in the blp 35Spro:PHYB doubly homozygous line. Lower levels of both transcripts and proteins of PHYB suggested that introduction of the chimeric gene interfered with the stability of PHYB transcripts. Our results highlight that overexpression mutagenesis facilitates functional genomics to decipher a function of Arabidopsis genome.

Plant breeding requires genetic diversity of useful traits for crop improvement. EMS-induced mutation is practiced to generate mutations at loci regulating economically important traits and/or to knock out the genes to elucidate their functions. The present study was aimed to induce mutations in a Korean local land race Capsicum annuum ‘Yuwol-cho’. This accession is pungent and also has advantage to mature early. A total of about 1,500 M2 families were screened and three non-pungent mutants were identified and crossed with wild type ‘Yuwol-cho’. After phenotyping of F2 population for pungency, MutMap approach will be used to identify the genes controlling the pungency in mutants. In addition to this, another C. annuum accession “Micro-Pep” was used to develop a mutant population. Micro-Pep is a small, pungent pepper generally used as ornamental purpose. Having compact growth habit, and small size, it has advantage to handle and utilize easily in mutation study and molecular research. On the basis of preliminary experiment 1.3% of mutagen was used for treatment of pepper seeds and 30% less germination percentage was observed in EMS treated seeds in comparison to control seeds. A total of 4,674 M1 plants are grown under greenhouse condition and M2 population will be studied for characterization of phenotypic variation including fruit color and pungency. Newly constructed mutant populations will be valuable assets for identification of functional genes and molecular breeding of pepper.