Cloning and Expression Analysis of a Brassinosteroid Biosynthetic Enzyme Gene, GhDWF1, from Cotton （Gossypium hirsuturm L.）

Brassinosteroids （BRs） are an important class of plant steroidal hormones that are essential in a wide variety of physiological processes. To determine the effects of BRs on the development of cotton fibers, through screening cotton fiber EST database and contigging the candidate ESTs, a key gene （GhDWF1） involved in the upstream biosynthetic pathway of BRs was cloned from developing fibers of upland cotton （Gossypium hirsutum L.） cv. Xuzhou 142. The full length of the cloned cDNA is 1 849 bp, including a 37 bp 5＇-untranslated region, an ORF of 1 692 bp, and a 120 bp 3＇-untranslated region. The cDNA encodes a polypeptide of 563 amino acid residues with a predicted molecular mass of 65 kD. The deduced amino acid sequence has high homology with the BR biosynthetic enzyme, DWARF1/DIMINUTO, from rice, maize, pea, tomato, and Arabidopsis. Furthermore, the typical conserved structures, such as the transmembrane domain, the FAD- dependent oxidase domain, and the FAD-binding site, are present in the GhDWF1 protein. The Southern blot indicated that the GhDWF1 gene is a single copy in upland cotton genome. RT-PCR analysis revealed that the highest level of GhDWF1 expression was detected in 0 DPA （day post anthesis） ovule （with fibers） while the lowest level was observed in cotyledon. The GhDWF1 gene presents high expression levels in root, young stem, and fiber, especially, at the fiber developmental stage of secondary cell wall accumulation. Moreover, the expression level was higher in ovules （with fibers） of wildtype （Xuzhou 142） than in ovules of fuzzless-lintless mutant at the same developmental stages （0 and 4 DPA）. The results suggest that the GhDWF1 gene plays a crucial role in fiber development.     

Susceptible time window and endurable duration of cotton fiber development to high temperature stress

The development of the cotton fiber is very sensitive to temperature variation, and high temperature stress often causes reduced fiber yield and fiber quality. Short-term high temperature stress often occurs during cotton production, but little is known about the specific timing and duration of stress that affects fiber development. To make this clear, pot experiments were carried in 2014 and 2015 in a climate chamber using cotton cultivars HY370WR(less sensitive variety) and Sumian 15(heat sensitive variety), which present different temperature sensitivities. Changes of the most important fiber quality indices(i.e., fiber length, fiber strength and marcironaire) and three very important fiber development components(i.e., cellulose, sucrose and callose) were analyzed to define the time window and critical duration to the high temperature stress at 34°C(max38°C/min30°C). When developing bolls were subjected to 5 days of high temperature stress at different days post-anthesis(DPA), the changes(Δ%) of fiber length, strength and micronire, as a function of imposed time followed square polynomial eq. as y=a+bx+cx~2, and the time around 15 DPA was the most sensitive period for fiber quality development in response to heat stress. When 15 DPA bolls were heat-stressed for different durations(2, 3, 4, 5, 6, 7 days), the changes(Δ%) of fiber length, strength and micronire, as a function of stress duration followed logistic equations y=A_1-A_2/1+(x/x_0)~p+A_2. Referred to that 5, 10 and 15% are usually used as criteria to decide whether techniques are effective or changes are significant in crop culture practice and reguard to the fiber quality indices change range, we suggested that 5% changes of the major fiber quality indices(fiber length, fiber strength and micronaire) and 10% changes of fiber development components(cellulose, sucrose and callose) could be taken as criteria to judge whether fiber development and fiber quality have been significantly affected by high temperature stress. The key time window for cotton fiber development in response to the high temperature stress was 13–19 DPA, and the critical duration was about 5 days.     

Developmental and hormonal regulation of fiber quality in two natural-colored cotton cultivars

Cotton cultivars with brown(Xiangcaimian 2), green(Wanmian 39) and white(Sumian 9) fiber were investigated to study fiber developmental characteristics of natural-colored cotton and the effect of hormones on fiber quality at different stages after anthesis. Fiber lengths of both natural-colored cottons were lower than the white-fibered control, with brown-fibered cotton longer than green. Fiber strength, micronaire and maturation of natural-colored cotton were also lower than the control. The shorter fiber of the green cultivar was due to slower growth during 10 to 30 days post-anthesis(DPA). Likewise, the lower fiber strength, micronaire and maturation of natured-colored cotton were also due to slower growth during this pivotal stage. Indole-3-acetic acid(IAA) content at 10 DPA, and abscisic acid(ABA) content at 30 to 40 DPA were lower in the fibers of the natural-colored than that of the white-fibered cotton. After applying 20 mg L~(–1) gibberellic acid(GA3), the IAA content at 20 DPA in the brown and green-fibered cottons increased by 51.07 and 64.33%, fiber ABA content increased by 38.96 and 24.40%, and fiber length increased by 8.13 and 13.96%, respectively. Fiber strength, micronaire and maturation were also enhanced at boll opening stage. Those results suggest that the level of endogenous hormones affect fiber quality. Application of external hormones can increase hormone content in natural-colored cotton fiber, improving its quality.     

Cloning and Expressing of a Gene Encoding Cytosolic Copper/Zinc Superoxide Dismutase in the Upland Cotton

In this study, a gene encoding a superoxide dismutase （SOD） was cloned from senescent leaves of cotton （Gossypium hirsutum）, and its expressing profile was analyzed. The gene was cloned by rapid amplification of cDNA ends （RACE） method. Northern blotting was used to show the profile of the gene expression, and the enzyme activity was mensurated by NBT deoxidization method in different growth periods. The full length of a gene of cytosolic copper/zinc superoxide dismutase （Cu/Zn-SOD） was isolated from cotton （GenBank Accession Number： DQ445093）. The sequence of cDNA contained 682 bp, the opening reading frame 456 bp, and encoded polypeptide 152 amino acids with the predicted molecular mass of 15.03 kD and theoretical pI of 6.09. The amino acid sequence was similar with the other plants from 82 to 87%. Southern blotting showed that the gene had different number of copies in different cotton species. Northern blotting suggested that the gene had different expression in different tissues and development stages. The enzyme activity was the highest in peak flowering stage. The cotton cytosolic （Cu/Zn-SOD） had lower copies in the upland cotton. The copper/zinc superoxide dismutase mRNA expressing level showed regular changing in the whole development stages; it was lower in the former stages, higher in latter stages and the highest at the peak flowering stage. The curve of the copper/zinc superoxide dismutase mRNA expressing level was consistent with that of the Cu/Zn-SOD enzyme activity. The copper/zinc superoxide dismutase mRNA expressing levels of different organs showed that the gene was higher in the root, leaf, and lower in the flower.     

Response of Cytochrome P450 Expression to Maize Volatiles in Helicoverpa armigera (Hner)

The 7-ethoxycoumarin O-deethylase (ECOD) activities of cytochrome P450s and differential expression of six cytochrome P450 genes induced by the volatiles from both damaged and undamaged maize plants were investigated in the cotton bollworm, Helicoverpa armigera (Hner). The ECOD activity changed with time of exposure to maize volatiles. At 36 h after cotton bollworm larvae exposure to maize volatiles, the ECOD activities in cotton bollworm damaged and artificially damaged groups were 2.36 and 4.53 times higher than the control group respectively. The relative expression levels of CYP4S1, CYP6B2 and CYP6B7 in the cotton bollworm were significantly increased in artificially damaged plant group, which was 2.93, 5.09 and 10.66 times higher than that in the control group, respectively. The expression levels of CYP6B2, CYP6B6, CYP9A12, and CYP9A14 were much lower in the larvae exposure to volatiles from both healthy and pest damaged maize seedlings than in the control group at 12 h after larvae exposure to maize volatiles. For the cotton bollworm damaged maize group, the expression of CYP4S1 and CYP9A14 increased.     

Progress in understanding hormonal regulation during the postembryonic development of Helicoverpa armigera

Lepidoptera, with 0.16 million species, is the second largest order of insecta. This order includes silk worms, butterflies, and many agricultural pests. The cotton bollworm, Helicoverpa armigera(Hübner) is one of the pests in Lepidoptera that seriously harms cotton plant and other crops in China and other countries. This pest develops resistance to chemical insecticides rapidly. Bacillus thuringiensis(Bt) transgenic cotton plants are developed to control H. armigera population in the field. However, during the past years, the rapid evolution of Bt toxin resistance is observed in H. armigera in transgenic cotton fields. New approaches for the development of new environmentally friendly insecticides to control H. armigera have become necessary, and the molecular mechanisms underlying the development and physiological processes of this species need to be further understood. Considerable progress in the study of H. armigera development and physiology has been achieved in the last decade. This mini-review summarizes the main findings on the molecular mechanisms of hormonal regulation of the development of H. armigera to present new target genes for developing new approaches to control the pest.     

Differential Gene Expression Between Hybrids and Their Parents During the Four Crucial Stages of Cotton Growth and Development

The study aims to clarify the differential gene expression between cotton hybrids and their parents in order to better understand the molecular basis of cotton heterosis. The research focused on cotton heterotic and lower heterotic hybrids and their parents during the four crucial stages, which were analyzed using a differential display technique. The results indicated that there were both quantitative and qualitative differences in gene expression amongst them. The quantitative differences include over- and under-expression of parental genes and the dominant expression of highly-expressed parental genes in hybrids. In contrast, the qualitative differences are the following： （i） Bands were observed in both parents but not in the F1 hybrid （BPnF1）; （ii） bands occurred in either of the parents but not in the F1 hybrid （UPnF1）; （iii） bands presented only in the F1 hybrid but not in either of the parents （UF1nP）; and （iv） bands were detected in either of the parents and the F1 hybrid （UPF1）. Overall, the major differences of gene expression occurred in the qualitative level and four related differential patterns were observed. Furthermore, the amount of differential patterns during the flowering stage was relatively higher than those of other stages. At this juncture, both the amount of hybrid-specific expression patterns at flowering stage and the silenced expression patterns at boll-forming stage in highly heterotic hybrids were found higher than those in the lower heterotic ones. It was concluded that significant differences of gene expression in leaves were present between cotton hybrid and its parents during the whole growing stages. Hence, these differences might be responsible for the observed cotton heterosis.     

Effects of Increased Night Temperature on Cellulose Synthesis and the Activity of Sucrose Metabolism Enzymes in Cotton Fiber

Temperature is one of the key factors that influence cotton fiber synthesis at the late growth stage of cotton. In this paper, using two early-maturing cotton varieties as experimental materials, night temperature increase was stimulated in the field using far-infrared quartz tubes set in semi-mobile incubators and compared with the normal night temperatures (control) in order to investigate the effects of night temperature on the cotton fiber cellulose synthesis during secondary wall thickening. The results showed that the activity of sucrose synthase (SuSy) and sucrose phosphate synthase (SPS) quickly increased and remained constant during the development of cotton fiber, while the activity of acid invertase (AI) and alkaline invertase (NI) decreased, increased night temperatures prompted the rapid transformation of sugar, and all the available sucrose fully converted into cellulose. With night temperature increasing treatment, an increase in SuSy activity and concentration of sucrose indicate more sucrose converted into UDPG (uridin diphosphate-glucose) during the early and late stages of cotton fiber development. Furthermore, SPS activity and the increased concentration of fructose accelerated fructose degradation and reduced the inhibition of fructose to SuSy; maintaining higher value of allocation proportion of invertase and sucrose during the early development stages of cotton fiber, which was propitious to supply a greater carbon source and energy for cellulose synthesis. Therefore, the minimum temperature in the nightime was a major factor correlated with the activity of sucrose metabolism enzymes in cotton fiber. Consequently, soluble sugar transformation and cellulose accumulation were closely associated with the minimum night temperature.     

Regulatory Expression of Peroxisome Proliferator Activated Receptors Genes During Fatty Liver Formation in Geese

In order to investigate the expression pattern of peroxisome proliferator activated receptor （PPAR） genes before and after overfeeding, and estimate the effect of expressed PPAR levels on weights of fatty liver and abdominal fat in geese, the RT-PCR products of PPAR genes in heart, liver, spleen, lung, kidney, stomach, small intestine, brain, breast muscle, leg muscle, and abdominal fat were determined before and after overfeeding. RT-PCR was used to determine the expression levels of PPAR genes. Quantity one software was used to analyze absorbency, and the expression level of GAPDH gene was used as contrast. Expression levels of PPAR-α were relatively high in most of detected tissues but undetectable in abdominal fat tissue before overfeeding, and the level was evidently increased in lung, appeared in abdominal fat tissue, and reduced in the other tissues after overfeeding. Expressed PPAR-γ levels were relatively high in liver, spleen, lung, small intestine, and abdominal fat, and relatively low in the other tissues before overfeeding. Expressed PPAR-γ levels were enhanced in liver, spleen, lung, stomach, and kidney but decreased in abdominal fat and without obvious changes in the other tissues. Expression patterns of PPAR genes show tissue-specific manner. In addition, expression patterns of PPAR-α are different from PPAR-γ after overfeeding. It might suggest that different functions of PPAR subtypes are responsive to overfeeding.     

Gene Cloning and Tissue-Specific Expression of G Protein β Subunit in Microplitis mediator (Hymenoptera: Braconidae)

A gene encoding a novel G protein β subunit of β1 subclass, GβMmed was isolated from Microplitis mediator (Hymenoptera: Braconidae). The full-length sequence of GβMmed is 1 119 bp, the cDNA contains a 1 023 bp open reading frame that encodes a protein with 340 amino acids, and the predicted molecular weight of GβMmed is 37.23 kDa and isoelectric point is 5.86. By the quantitative real-time RT-PCR method, the tissue-specific expression and quantitative changes in the developmental expression profile of GβMmed were detected. It was found that GβMmed was abundantly expressed in M. mediator antennae, head (without antennae), thorax, abdomen, legs and the wings, and especially at high levels in abdomen. In antennae, expression varied through 1st day before emergence to 5-d-old adults, and had equal expression levels detected in females and males in total. In head, GβMmed expresses while initially high in females, and have another peaked in stage 4 and 1st day, in males showed a peak of GβMmed expression prior to emergence and relatively low levels after emergence. In female abdomen GβMmed expression levels have two peaks in stage 1 and the 5th d, but just have one peak in male abdomen in stage 1. In all other tissues expression was low and stable.     

Myofiber development during embryonic to neonatal development in duck breeds differing in muscle growth rates

Little is known about the muscle developmental patterns during embryonic to neonatal development in ducks.We investigated the developmental patterns in the lateral gastrocnemius muscles of Gaoyou and Jinding ducks differing in their muscle growth rates during the final stages of egg incubation and the first week after hatching.Expression of the MyoD gene was quantified by quantitative real-time PCR(qRT-PCR).The average cross-sectional area and diameter of the fibers increased from embryonic day 21(E21),peaking at E27,and then declining slightly 7 d after hatching.The density of the fibers decreased initially but increased after hatching in both breeds and sexes.The within-breed variation in muscle fiber-type composition was greater than the average variation between the breeds.Overall,the percentage of type Ⅰ fibers increased and that of type lIb fibers decreased consistently.However,the percentage of type lla fibers was almost constant as development proceeded in both duck breeds.The profiles of MyoD mRNA expression were similar in both breeds,and a significantly positive relationship was observed between the expression of MyoD and the percentage of type lIb fibers.This study firstly revealed the characteristics of duck muscle development and differences between the two breeds differing in growth rates.Moreover,type lIb fibers might convert to type Ⅰ fibers in the lateral gastrocnemius,while MyoD may potentially function in controlling the muscle fiber phenotype during the secondary myogenesis of muscle development.     

Evaluation on the High Yielding,Stability and Comprehensive Performances of Check Cultivars in National Cotton Regional Trials in the Yangtze River Valley

[Objective] This study was to analyze the high yielding, stability, adaptabil-ity and main characteristics of check cultivars in national cotton regional trials in the Yangtze River Val ey in 2000-2013, with the aim to provide theoretical basis for the reasonable selection of check cultivars for cotton regional trials. [Method] GGE biplot analysis method was used to carry out the intensive analysis and comprehensive comparisons on the performances and differences of the 9 major characters of check cultivars, namely, lint cotton yield , stability, and other main traits including bol weight, bol number, lint percentage, fiber length, fiber strength, micronaire val-ue, plant height and seed index. [Result] Ezamian 10 was suitable in the majority of cotton planting regions in the Yangtze River Val ey, with the best high yielding abili-ty and perfect yield stability. The yielding ability of Xiangzamian 8 ranked the sec-ond, but its stability was the poorest. Xiangzamian 8 was of the specific adaptive cultivar with relatively good performance in high yielding but the poorest stability. The suitable planting areas of Xiangzamian 8 were limited to the cotton planting re-gion around Dongting Lake in Hunan province, Poyang Lake in Jiangxi Province, the Coastal region in Jiangsu Province and Wuhan district in Hubei Province. Xi-angzamian 2 performanced poorer in both yielding and stability, while Simian 3 ranked first in stability but the last in yielding ability. The dominant character of Xi-angzamian 8 and Xiangzamian 2 were high cotton plants and big bol s. Simian 3 performanced super in lint percentage and fiber length. Ezamian 10 was strong in cotton bol formation ability, lint cotton yielding, fiber strength and seed size, as wel as high micronaire value. [Conclusion] The outstanding performance of Ezamian 10 in high and stable yield in the Yangtze River Val ey in recent years has effectively promoted the high yielding ability of candidate varieties in cotton regional trials in the area.     

Isolation and Characterization of Ell Gene Promoter from Tomato

A fragment of 2000 bp upstream sequence of Ell clone was amplified from genomic DNA of the tomato cultivar Zhongshu- 5. Sequence analysis showed that the upstream contains the regulatory elements： TATA box （-29 - -22）, CAAT box （-193 - -189）, wound, and drought response elements. Expression vectors of Ell promoter gus fusion were constructed with the promoters of 1 200 and 2 000 bp regions, respectively. Transgenic tomato plants were obtained through Agrobacteriummediated transformation. Histochemical analysis of GUS activity in various tissues showed that the two promoters were able to direct fruit-specific gene expression. The expression driven by promoter of 2 000 bp upstream fragment could increase GUS activity with the maturation of tomato fruits. The promoter of -1 200 bp fragment could direct gus gene expression in fruits with the inductions of drought and wounding. The regulatory region for fruit-specificity was probably located in the region of 1 200 bp of 5′-flanking sequence and some positive regulatory elements or enhancers may exist in the region from -1 200 to -2000 bp.