Observation of fiber ultrastructure of Ligon lintless mutant in upland cotton during fiber elongation

Lintless mutant is a super-short fiber mutanl in upland cotton only 4-8 mm in fiber length and also named Ligon cotton controlled by one dominant gene Li1. Fiber ultrastructure of the mutant (Li1) and its its wild (li1) in siti and in vitro was observed under an electron microscope to understand its cytological characteristics during the fiber cell elongation. The resulls showed that the mutant fiber in situ had thinner cytoplasm, more small vacuoles, less mitochondria, Golgi apparatus and endoplasmic reticula, and there were more starch granules which were free or packed in the amyloplast beside the cell wall than thai of wild type. It was indicated that scarcity of functional organelles and disability of transformation from starch to sugar might be associated with the tact that the mutant fiber cell was aborted too early to elongate into normal length. Mutant ovule in some media containing GA3 could produce a kind of huge callus that grew faster than normal ovules. The callus was covered with many white, loose, and semitransparent fiber-like cells that apt lo get off from ovule. These fiber-like cells were multicellular fibers generated by cell division and had black dots just like pigment glands in the stem and leaf of cotton. There were lots of micro-tubes beside cytoplasm membrane uf the multiecllular fiber, which were thought to be primary preparation for second wall deposition of multicellular fiber. It was indicated that GA3 might induce the expression of gene(s) that kept inactive in the field condition and then stimulate the original fiber cell in vitro to undergo divisionagain.     

Construction and characterization of partiallyntrC-deleted mutants inAlcaligenes faecalis

To study the effect of ntrC gene product on the expression and regulation of other important nitrogen-fixing genes in Alcaligenes faecalis, partially ntrC-deleted mutants of A. faecalis have been generated. To start with, the ntrC gene of A. faecalis was cloned into a suicide plasmid pSUP202 to create a recombinant plasmid pSUM1. The ntrC gene in pSUM1 was then replaced by a lacZ-Km^r fragment resulted in the generation of a plasmid pSUM2. The lacZ fragment in pSUM2 was further removed and a plasmid pSUM3 produced. As a second step, the plasmid pSUM2 or pSUM3 was introduced into the wild type of A. faecalis A1501 by conjugation and two partially ntrC-deleted mutants A15CM1 (ntrC∷lacZ) and A15CM2 (ntrC ^- ) were obtained. To understand the regulatory effect of the NtrC on the expression of nifH and nifA, a nifH-lacZ gene or a nifH-lacZ gene was introduced into the ntrC^- mutant by conjugation. The results indicated that: (ⅰ) although the ntrC^-mutant was nif ⁺ , its nitrogen fixation activity was only 20% that of the wild type; (ⅱ) the ntrC^- mutant failed to grow on the medium containing nitrate as a sole nitrogen source; (ⅲ) the regulation of ntrC gene expression did not require its own product; (ⅳ) the expression of nifH in A . faecalis was positively regulated by the ntrC. Deletion of the ntrC resulted in the reduction of nifH expression or even totally inactivated nitrogen fixation; (ⅴ) there was no obvious influence on the expression of nifA in A. faecalis if the ntrC gene was deleted.     

Effects ofPKCα antisense RNA on human breast cancer cell proliferation and expression ofcyclinD1 andCDK4

The role of PKCα in human breast cancer cell proliferation and expression ofcyclinD1 andCDK4 has been investigated using inhibition ofPKCα expression by its antisense RNA. WhenPKCα expression was inhibited the rate of cell proliferation decreased apparently and the levels ofcyclinD1 andCDK4 mRNA were lower than the control. The results showed thatPKCα, a key member of signal transduction system, played an important role in human breast cancer cell proliferation and had a close relationship with expression ofcyclinD1 andCDK4 which control start of cell cycle.     

Testing relative evolutionary rates and estimating divergence times among six genera of Rhizophoraceae using cpDNA and nrDNA sequences

The molecular phylogenetic trees of 10 species representing 6 genera of the family Rhizophoraceae have been constructed using the sequences of chloroplast genesmatK andrbcL as well as the ITS regions of nuclear ribosomal DNA. Relative-rate tests between lineages in these phylogenetic trees have been performed. On the basis of the results of the relative-rate tests and related molecular evolutionary rate data, the divergence times between the lineages are estimated as follows: (i) the first divergence time in these genera is 132.25 million years ago (mya); (ii) the average divergence time between two tribes, i.e. inland Legnotideae (exceptCarallia brachiata) and mangrove Rhizophoreae, is 64.13 mya; and (iii) the average divergence time between two inland species,C. garciniaefolia andC. pectinifolia, is 19.92 mya.     

Genetic analysis and gene mapping of a narrow leaf mutant in rice ( Oryza sativa L.)

A narrow leaf mutant was obtained after T-DNA transformation conducted on a rice variety Zhonghua 11. Several abnormal morphological characteristics, including semi-dwarf, delayed flowering time, narrow and inward rolling leaves, and lower seed-setting, were observed. The rate of net photosynthesis (under saturate light) of flag leaves in the mutant was significantly lower than that of the wild type. Moreover, the leaf transpiration rate and stomatal conductance in the mutant flag leaf were lower than those of the wild type at the grain filling stage. It was found that the mutant phenotype was not caused by the T-DNA insertion. Genetic analysis showed that the mutant was controlled by a single recessive gene, designated as nal3(t). A genetic linkage map was constructed using a large F₂ mapping population derived from a cross between nal3(t) and an indica variety Longtefu B with 6 polymorphic markers on chromosome 12 identified from 366 SSR markers by the BAS method. Gene nal3(t) was mapped between the markers RM7018 and RM3331. Fine mapping of nal3(t) locus was conducted with 22 newly developed STS markers based on the sequence diversity around the region harboring nal3(t) between Nipponbare and 93–11, and nal3(t) was finally mapped to a 136-kb region between the STS markers NS10 and RH12-8. Supported by National High Technology Research and Development Program of China (863 Program) (Grant No. 2006AA10A102), National Natural Science Foundation of China (Grant No. 30600349) and Natural Science Foundation of Zhejiang Province (Grant No. Y306149)     

Reduced Na+ and K+ permeability of K+ channel in plasma membrane isolated from roots of salt-tolerant mutant of wheat

The Na⁺ and K⁺ permeability of K⁺ channel in plasma membrane, isolated from roots of the salt-tolerant mutant of wheat, was lower than that of wild type in 100 mmol/L KCl and NaCl solution. The opening frequency of K⁺ channel of the mutant reduced more significantly than that of wild type in two kinds of solution mentioned above. It is assumed that the reduction of opening frequency mainly contributes to the Na⁺ and K⁺ permeability of K⁺ channel of the mutant. The electric conductance of single-channel of the mutant was similar to that of wild type and the main difference between them was exhibited as the opening frequency. Their K⁺/Ka⁺ selectivity of K⁺ channel had no significant difference. The K⁺/Na⁺ selectivity of the mutant and wild type was 3.35 and 3.18 respectively.     

Localization of muscarinic acetylcholine receptor in plant guard cells

Acetylcholine (ACh), as an important neurotransmitter in animals, also plays a significant role in various kinds of physiological functions in plants. But relatively little is known about its receptors in plants. A green fluorescence BODIPY FL-labeled ABT, which is a high affinity ligand of muscarinic acetylcholine receptor (mAChR), was used to localize mAChR in plant guard cells. InVicia faba L. andPisum sativum L., mAChR was found both on the plasma membrane of guard cells. mAChR may also be distributed on guard cell chloroplast membrane ofVicia faba L. The evidence that mAChR localizes in the guard cells provides a new possible signal transduction pathway in ACh mediated stomata movement.     

A taxonomic investigation of variation withinCissus quadrangularis L. (Vitaceae) in Kenya

Using information from literature as well as records of localities on herbarium specimen labels at the East African Herbarium Nairobi (EA), materials ofCissus quadrangularis L.,C. cactiformis Gilg. andC. quinquangularis Chiov. were collected and new localities were sought. On the basis of the nature of their stem angles, the specimens ofC. quadrangularis were sorted into two variants, A and B. Variant A has smooth stem angles while variant B has rough stem angles. The authors have proposed that variant B should be recognized as a new variety withinC. quadrangularis. Variant A is the type variety ofC. quadrangularis. A map showing the distribution of the two variants in Kenya has been prepared. The rough-edged variant was found to be absent from the low-lying semi-arid regions of northern Kenya. The variants were found occurring in distinct, separate populations but without separate geographical patterns. The results of t-test show no significant difference (p=0.05) between the means of measurements of dimensions of various organs in the two variants. The stem angles of specimens of the two variants were studied under scanning electron microscope (SEM). This showed a clear morphological difference between the two variants. The results of the investigation support the hypothesis that the variation withinC. quadrangularis represents distinct kinds that can be recognized as taxa at infra-specific level. Foundation item: Supported by the China Ministry of Education Fund for University Staff Development in Key Subject Areas and Kenyatta University. Biography: Robert Wahiti. Gituru (1967-), male, Kenya, Ph. D. candidate, research direction: plant biosystematics.     

Interspecific hybridization ofHelicoverpa armigera andH. assulta (Lepidoptera: Noctuidae)

Reciprocal hybridization between Helicoverpa armigera (Hübner) and H. assulta Guenee followed by backcrossing of the hybrids (F₁) with H. armigera produced backcross (BC) lines consisting of fertile females and males. The F₁ of H. armigera female and H. assulta male had only male, no female sex. In this case Haldane’s rule applies, and therefore it is proved that the sex chromosomes of Helicoverpa species are of ZW type, and the female is the heterozygous sex. This hybrid also showed significant heterosis with the heaviest pupal weight, and when it was backcrossed with H. armigera female, the sex ratio of the BC offspring was distorted as 1∶4. The potential utilization of this hybrid in genetic controlling H. armigera was finally discussed.     

Variation induced by DNA rearrangement in a transgenicBt+CpTI cotton strain

In the development of transgenicBt + CpTI cotton cultivars, one male and female sterile mutant has been found in a homozygous T₄ strain in our laboratory. The mutant plant, as well as its leaves, buds and flowers, is only 1/2–1/3 as large as that of the wild transgenicBt + CpTI bivalant cotton plants. Cytological observation found that the chromosome number of the mutant is 2n = 52; however, there are 4–8 univalents observed in meiosis I of pollen mother cells. Laboratory bioassay indicated that the mutant was highly resistant to bollworm as the wild plants. PCR amplification revealed thatBt andCpTI genes in the mutant were still intactly inserted. However, small deletion of flanked area had been observed in the mutant by Southern blotting analysis. So it is proposed that the mutant phenotype might result from either the DNA deletion or T-DNA transferring in plant genome. No such report has been presented that the rearrangement of chromosome structure in a homozygous transgenic line occurred. Further analysis is ongoing.     

利用CRISPR /Cas9技术快速创制香型“秀水134”水稻

以目前上海市主栽的高产常规水稻"秀水134"为材料,利用CRISPR/Cas9技术成功敲除甜菜碱醛脱氢酶2基因,获得了两种类型纯合突变体植株.采用表达载体特异性结合的引物检测T_1代转基因植株,成功获得6株不携带载体骨架的转基因植株.定量PCR分析显示,突变体植株甜菜碱醛脱氢酶2基因表达量极显著低于野生型对照(p0.01),但突变体植株成熟种子香味物质2-乙酰-1-吡咯啉(2AP)含量极显著高于野生型对照(p0.01).比较野生型对照与突变体植株的主要农艺性状和产量性状,两者间都没有显著差异(p0.05).本研究可为加快高产香型水稻在上海及周边地区的推广应用,以及为今后利用CRISPR/Cas9技术快速培育其他高产香型水稻新品种研究奠定基础.     

Cloning of fiber-specific cDNAs and their structural variations in 4 fiber mutants

A mRNA preferentially expressed in cotton fiber was cloned from fiber total RNA of normal upland cotton TM-1 (wild-type) by using RT-PCR and corresponding cDNA (signed as TM-E6) was sequenced. TM-E6 gene had no intron and contained an open reading frame of 771 bp long, and might encode a peptide of 246 amino acids. Other 4 genes, Fl-E6, Li-E6, N-E6 and Bl-E6, which were homologous to TM-E6 gene, were also isolated from 4 fiber mutants of Fiberless Xu-zhou 142, Ligon lintless, Naked seed and Brown lint, respectively. Sequence analysis of each of these mutant genes revealed many variations in structure and nucleotide composition of gene when compared with the sequence of TM-E6 gene. (ⅰ) There was a changeable repetitive segment in which GGCTCA (Gly-Ser) was repeated 3—5 times between the 82nd and the 93rd codons in different mutant genes. Since the change of Gly-Ser repetitive segment occurred not only in the mutants but also in the wild-type cotton, the repeat frequency might not be associated with the mutation of fiber characteristics. (ⅱ) Among the 4 mutant genes, the percentage of changed codons was 7.05% in Fl-E6, 4.98% in Li-E6, and 4.15% in N-E6 and Bl-E6. It seems that the percentage of changed codons in E6 sequence was positively correlated to the degree of fiber morphological variation. (ⅲ) E6 polypeptides of two long-fiberless mutants (Fiberless Xuzhou 142 and Ligon lintless) contained high similar (99.4%) variation in the region of 1—174 amino acids from N-terminus, and those of short-fiberless mutants (Fiberless Xuzhou and naked seed) revealed identical variation in the region of 116th—220th amino acids. It also seems that there was a parallel relation between E6 protein variation and fiber phenotype mutation. (ⅳ) Li-E6 and Bl-E6 genes also expressed at low level in seed coat besides at high level in fiber.     

Influence of site-directed mutation of cytochrome b5 Phe35 on the protein’s structure and properties

Kinetic studies of the heme dissociation from the wild type and Phe35Tyr, Phe35Leu mutants of bovine liver microsomal ferricytochrome b 5 indicate that the oxidized Phe35Tyr mutant is more stable towards denaturant than wild type but Phe35Leu mutant proceeded with a different mechanism compared with wild type cytochrome b 5 and Phe35Tyr mutant protein. Because of the decrease of side chain volume in Phe35Leu mutant, a cavity produced in the interior of the protein may offer a channel for urea molecule to enter the hydrophobic pocket. When urea concentration is larger than 5 mol/L, the urea molecule may compete to coordinate the iron of heme with His39, that results in sharp increase of the rate of heme dissociation. The interaction between cytochrome b 5 and cytochrom c demonstrated that a 1:1 protein complex was formed between the two proteins. The binding constants of cytochrome b 5 with cytochrome c are: wild type K A=4.2(±0.01)×10 6(mol/L) -1 , Phe35Tyr K A=3.7(±0.01)×10 6(mol/L) -1 and Phe35Leu K A=4.7(±0.01)×10 6(mol/L) -1 respectively ( I =1 m mol/L, pH 7.0 soldium phosphate buffer, 25℃). These results clearly show that the mutation at Phe35 has no influence on the binding of cytochrome b 5 with cytochrome c and that the hydrophilic patch residues are not involved in the binding of cytochrome b 5 and cytochrome c.     

A necessary and sufficient condition on robust SPR stabilizability for low degree systems

For low degree systems (n≤3), it is verified that all convex combination of α(s) and b (s) keeping Hurwitzness is a necessary and sufficient condition for the existence of c (s) such that both c(s)/α(s) and c(s)/b(s) areSPR.     

Genetic analysis and gene fine mapping of a rolling leaf mutant (<Emphasis Type="Italic">rl</Emphasis><Subscript><Emphasis Type="Italic">11(t)</Emphasis></Subscript>) in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The exploration of new genes controlling rice leaf shape is an important foundation for rice functional genomics and plant archi-tecture improvement. In the present study, we identified a rolling leaf mutant from indica variety Yuefeng B, named rl_11(t), which exhibited reduced plant height, rolling and narrow leaves. Leaves in rl_11(t) mutant showed abnormal number and morphology of veins compared with those in wild type plants. In addition, rl_11(t) mutant was less sensitive to the inhibitory effect of auxin than the wild type. Genetic analysis suggested that the mutant was controlled by a single recessive gene. Gene Rl_11(t) was initially mapped between SSR markers RM6089 and RM124 on chromosome 4. Thirty-two new STS markers around the Rl_11(t) region were developed for fine mapping. A physical map encompassing the Rl_11(t) locus was constructed and the target gene was finally delimited to a 31.6 kb window between STS4-25 and STS4-26 on BAC AL606645. This provides useful information for cloning of Rl_11(t) gene.     

Kinetic studies on the irreversible inhibition of restriction endonucleasePst I by site-specific inhibitors

The irreversible modifying effects onPst I of several inhibitors have been studied with the irreversible inhibition kinetic theory of single substrate reaction provided by Tsou, C. L. Pyridoxal phosphate (PLP), p-chloromercuribenzoic acid (PCMB), diisopropyl fluorophosphate (DEP), 2,3-diacetyl (DAC) and N-ethyl-5-phenylisoxazoliun-3′-sulfonate (woodward's reagent K, WRK) modify the lysine, cysine, serteine, arginine and carboxyl groups of the protein molecule respectively. These five inhibitors have been found to inhibit both the prime activity and star activity ofPst I. Used with the irreversible inhibition theory, the apparent inhibition rate constant,A and the microcosmic inhibition rate constants,k ₊₀ andk′ ₊₀ of every inhibitor were calculated. We also found that their inhibition effects belong to the noncompetitive irreversible inhibition. Results show that among the groups to be modified, some have nothing to do with the combination with the substrate, and some may have, but any of them isn't the only factor involved in the specific binding. Despite all this, they may take part in the catalysis of enzyme or have important effects on maintaining the active structure of enzyme molecules. Furthermore, serine and arginine residues are related to the alteration ofPst I conformation and then influence the ability ofPst I recognizing and incising DNA specifically. Foundation item: Supported by the Research Fund for the Doctoral Program of Higher Education (1999048601) Biography: Zou Guo-lin (1947-), male, Professor, research direction: enzymology.     

Mutagenesis of Ser24 of Cytochrome b559 α Subunit Affects PSII Acitivies in Chlamydomonas reinhardtii

In order to study the functions of cytochrome b559 (Cyt b559) in photosystem two (PSII) activity, mutant S24F of Chlamydomonas reinhardtii was constructed using site directed mutagenesis, in which Serine24 (Ser24) locating downstream of Histidine23 (His23) in α subunit of Cyt b559 was replaced by Phenylalanine (Phe). Physiological and biochemical analysis showed that mutant S24F could be grown photoautotrophically or photoheterotrophically. However, their growth rate was slower either on HSM or TAP medium than that of the control; Analysis of PSII activity revealed that its oxygen evolution was about 71% of wild type (WT); The Photochemical efficiency of PSII (Fv/Fm) of S24F was reduced 0.23 compared with WT; S24F was more sensitive to strong light irradiance than the wild type; Furthermore, SDS-PAGE and Western-blotting analysis indicated that the expression levels of α subunit of Cyt b559, LHCII and PsbO of S24F were a little less than those of the wild type. Overall, these data suggests that Ser24 plays a significant role in making Cyt b559 structure maintain PSII complex activity of oxygen evolution although it is not directly bound to heme group.     

An on-line multi-parameter analyzing optical biosensor for real-time and non-invasive monitoring of plant stress responses in vivo

Photosynthetic dysfunction and reactive oxygen species （ROS） production are the common features of plant stress responses. Based on quantitative measurement of ROS production and delayed fluorescence （DF） emission, which is an excellent marker for evaluating photosynthesis, an on-line multiparameter analyzing optical biosensor for detecting plant stress responses was developed. Performances of the proposed biosensor were tested in the wild type （WT） Arabidopsis and heat shock protein （Hsp） 101 T-DNA knockout mutant （hsp101） plants with different thermotolerance. Results demonstrated that DF intensity correlates with net photosynthesis rate （Pn） in response to elevated temperature in both the WT Arabidopsis and hsp101 mutant plants. The light response characteristics and the recovery dynamics of the DF intensity were also in line with those of Pn in both the WT Arabidopsis and hsp101 mutant plants after heat stress （HS, 40℃ for 30 min）, respectively. In all experiments discussed above, the hsp101 plant showed the worse photosynthetic performance than the WT plant. Moreover, after HS, more ROS production in the hsp101 mutant than in WT Arabidopsis, which was found to be mainly localized at chloroplasts, could be directly detected by using the proposed biosensor. In addition, the hsp101 mutant showed severer chloroplasts alterations than the WT plant within the first 1 h of recovery following HS. Nevertheless, pre-infiltration with catalase （CAT） reduced ROS production and prevented the declines of the DF intensity. Therefore, HS-caused declines of photosynthetic performance might be due to oxidative damage to photosynthetic organelle. To sum up, we conclude that Hsp101 plays an important role in preventing oxidative stress, and the proposed optical biosensor might be a powerful tool to determine plant stress responses and identify plant resistant difference.