Current Biotechnology

Functional Characterization of a Novel Chitinase Gene LcChi2 in Transgenic Rice
JIN Xue-bo1, 2, LIU Qing2, YIN Yue-jia2, JIN Yong-mei2, WANG Yun-peng2, YU Ying3, HAN Si-ping2*
2016, 6(2): 77-84. DOI: 10.3969/j.issn.2095-2341.2016.02.01
Abstract ( ) PDF (5527KB)( )

A new chitinase gene LcChi2 from Leymus chinensis showed enhanced resistance to fungal disease, when it was over-expressed in the dicotyledonous plant tobacco. Bioinformatics analysis suggested that LcChi2 encodes a type II chitinase classifying in the family 19. However, its resistance function to disease in the monocotyledon is unknown. In this study, we took Jijing 88, a widely grown Japonica rice cultivar in Jilin province, as the receptor plant of which the LcChi2 and herbicide screening marker gene Bar were transferred via Agrobacterium-mediated transformation. The transgenic rice at T1 stage was confirmed by PCR, RT-PCR and chitinase activity measurements, indicating that LcChi2 was integrated into the nuclear genome of rice and expressed effectively. The transgenic rice was artificially inoculated with the rice blast pathogens, showing higher resistance to the blast. Also, the transgenic rice exhibited herbicide resistance suggesting that the herbicide resistant gene Bar is functioning. Current results revealed that LcChi2 was able to improve the resistance to disease in the monocotyledons effectively, and might be used as a significant candidate disease-resistance gene for biotech breeding in the crops.

The Characteristics of Cd, Pb and As Accumulation in Rice Grain of Different Variety
DU Rui-ying, WEN Dian, ZHAO Di, ZHAO Pei-hua, PENG Li-jun, LI Lei, CHEN Yan, WANG Fu-hua*
2016, 6(2): 85-90. DOI: 10.3969/j.issn.2095-2341.2016.02.02
Abstract ( ) PDF (1056KB)( )

In order to study the difference of Cd, Zn and As accumulation in different cultivated variety rice. 316 rice cultivars varieties cultivated in a heavy metal polluted paddy field which located in a mine zone of northern Guangdong Province in 2014. The results showed that: The average values of Pb, As and Cd in traditional rice were as follows: 0.290 mg/kg, 0.060 mg/kg and 0.144 mg/kg. The contents of Pb, As and Cd in hybrid rice were as follows: 0.237 mg/kg, 0.044 mg/kg and 0.132 mg/kg. The content of heavy metals in traditional rice was higher than that in hybrid rice. The transfer ability of husk to rice was Cd>As>Pb. The strains of Yinzhan, Meixiangzhan and Shenliangyou had greater abilities to enrich Cd than other strains. The Shenyou strains had the weakest capacities to absorb Cd and Pb. The absorption of As and Cd in traditional rice was antagonistic, but the absorption of Pb and Cd in hybrid rice was synergistic. Therefore, according to the difference of heavy metal accumulation in different genotypes of rice, it is feasible to screen out the rice varieties of low accumulation of heavy metals.

Fusion Expression and Functional Analysis of Nicotiana tabacum Nt4CL and Polygonum cuspidatum PcSTS Gene
LIU Wen-bin1, GUO Hui-li2, LIU Huan1, HUANG Li-
2016, 6(2): 91-97. DOI: 10.3969/j.issn.2095-2341.2016.02.03
Abstract ( ) PDF (2154KB)( )

4-coumarate coenzyme A ligase (4CL) and stilbene synthase (STS) are two key final rate-limiting enzymes in the resveratrol phenylalanine metabolic synthesis. In this paper, Nicotiana tabacum 4CL and Polygonum cuspidatum PKS5 were fused together by overlap PCR with three amino acids to construct fusion gene Nt4CL-PcPKS5. The fusion gene was subcloned into pET30a, the recombinant plasmid pET30a-Nt4CL-PcPKS5 was analyzed through heterologous expression in Escherichia coli. The expression product was purified with Ni2+ affinity column and desalted through PD-10 column. The collected target fusion protein was used for enzymatic reaction in vitro. The enzymatic product analysis showed that the fusion protein showed the activities of both 4CL and STS. The final product was resveratrol. The optimum pH and reaction temperature were 6.5 and 45℃. In this study, a bifunctional fusion enzyme catalyzing resveratrol biosynthesis was obtained, which proved a basis for realizing industrial production of resveratrol.

Enzymatic Degradation of Ginsenoside Rb1 for Preparation of Compound K by Aspergillus niger sp. J7
GAO Juan1, ZHOU An-dong2, YUAN Ye1, ZHAO Tian-
2016, 6(2): 98-104. DOI: 10.3969/j.issn.2095-2341.2016.02.04
Abstract ( ) PDF (3017KB)( )

Minor ginsenoside Compound K (C-K) exhibited remarkable biological activities. In this study, nine fungi were isolated from soil, and their abilities of transform ginsenoside Rb1 for preparation of C-K were studied. A highly efficient ginsenoside Rb1-hydrolyzing fungus Aspergillus niger sp. J7 was screened. The strain converted ginsenoside Rb1 to C-K with the pathway of Rb1→Rd→F2→C-K. The conversion conditions of A. niger sp. J7 was optimized. Under the optimal conditions, ginsenoside Rb1 was converted to C-K completely. The transformation system was enlarged to 200 mL, in which ginsenoside Rb1 was completely converted into C-K after 60 h and the conversion rate was 74.7%. This fungus was expected to be the basis in bioactive C-K preparation.

The Acquisition and Bioinformatic Analysis of Sucrose Transporter Protein Gene PaSUC4 from Apricot
KANG Shuang1,2, HE Hong-xia2*, WANG Ming1*, GUO
2016, 6(2): 105-112. DOI: 10.3969/j.issn.2095-2341.2016.02.05
Abstract ( ) PDF (3911KB)( )

Sucrose is transported into fruit cells by sucrose transporters in fruit trees. The transport efficiency of sucrose transporters affects the sugar content and quality of fruits. Reverse transcript PCR technology was used in this study. Prunus armeniaca sucrose transport protein gene SUC4 (PaSUC4) was obtained from Jinong red apricot. The full cDNA was 2 480 bp according to the sequencing result. The codon region was 1 500 bp, encoding 499 amino acids. The protein molecular weight was 53.58 kDa, the soelectric point was 9.31. Results of similarity and phylogenetic analysis showed that the sucrose transporters we got had high similarity with that in Prunus (Rosaceae), speculating the function was also conserved. The expression level of PaSUC4 was highest in mature leaves and lowest in pistils. The characteristics of predicted protein had been analyzed by bioinformatics software. For further verification of the gene biological function, the expression vector had been constructed and transformed into tobacco plants. The research results was expected to provide reference for apricot breeding.

Population Dynamics of Bradysia odoriphaga and its Relationship with Temperature
HU Xia, BAI Yi-chuan, XU Wei-hong, ZOU De-yu, XU
2016, 6(2): 113-118. DOI: 10.3969/j.issn.2095-2341.2016.02.06
Abstract ( ) PDF (2487KB)( )

Bradysia odoriphaga is one of the most important insect pests of Chinese chives. In order to provide a theoretical basis for the prevention and control of Bradysia odoriphaga, we investigated anniversary dynamic of Bradysia odoriphaga in three cultivation model, such as solar greenhouse, plastic greenhouse and open. We investigated the annual population dynamics of Bradysia odoriphaga and analyzed the relationship between Bradysia odoriphaga population quantity and temperature, combined with temperature data. The results showed that there were three population peaks in spring, autumn and winter in solar greenhouse and open, respectively, and one corresponding lower amounts in summer; there were two population peaks in spring and autumn in plastic greenhouse, respectively, and two corresponding lower amounts in summer and winter. The lower population size in summer occurred in high temperature period. High-instar larva were dominant in the overwintering population of Bradysia odoriphaga. Analysis of correlation between population size and temperature showed that temperature was one of the key factors which decided Bradysia odoriphaga population numbers, and high temperature had significant inhibition effect on population growth.

Screening of New Chitinase Genes with Metagenomics
WANG Hai-dong1, HU Zhong2*
2016, 6(2): 119-124. DOI: 10.3969/j.issn.2095-2341.2016.02.07
Abstract ( ) PDF (2508KB)( )

In order to find new chitinease-encoding genes, we extracted metagenome from surface sediments of the Shantou Bay, and amplified 63 chitinase coding gene fragments using PCR-DGGE. Compared these fragments to NCBI records, the similarity ratios were from 41% to 97%. The most similarity ratios were below 70%, only 8 higher than 70%, and there were 18 species in NCBI chitinase records had the highest similarity ratios. 27 fragments had the highest similarity ratios to the chitinase sequence of Herpetosiphon aurantiacus ATCC 23779. 22 fragments had the highest similarity ratios to uncultured bacteria chitinase sequences which belonged to 9 different species, accounted for 34.9%. The results suggested that there were many different chitinase-coding genes in Shantou Bay sediments, most of them hadn’t yet been studied from now. The paper provided a biological resources for finding new chitinease-encoding genes.

Research on Extraction Process of Flavonoids From Pomelo Peel
LI Ge1, WANG Shan-shan1*, DING Wen-jun1, NIU
2016, 6(2): 125-129. DOI: 10.3969/j.issn.2095-2341.2016.02.08
Abstract ( ) PDF (1074KB)( )

Pomelo peel contains many kinds of active ingredients, such as pecitin, dietary fiber, essential oil, polysaccharide and flavonoids, which has high medicinal and edible value. Reasonable development, utilization and deep processing using pomelo peel as material, could not only reduce the environment pollution, but also create good economic and social benefits. This experiment extracted flavonoids from the pomelo peel using the ultrasonic method, and studied the influence factors in the process of pomelo peel flavonoids extraction. Through the single factor and orthogonal experiment method to study the optimal extraction technological parameter, results showed that the optimal parameters were as follows: ethanol concentration was 80%, ultrasonic treatment time was 30 min, soaking temperature was 60℃, solid to liquid ratio was 1:25, and the yield of flavonoid was 4.63%.

Progress and Prospects of Rolling Circle Amplification
SUN Ya-jun, WANG Liang, CAI Jun*
2016, 6(2): 130-136. DOI: 10.3969/j.issn.2095-2341.2016.02.09
Abstract ( ) PDF (1357KB)( )

The establishment of rolling circle amplification simulates the principle of self-replication of the ring-shaped pathogen DNA through the rolling circle model in nature, experiencing long-term scientific research and practical application, many breakthroughs have been achieved. This paper made a comprehensive summary on the latest research in recent years on rolling circle amplification(RCA), including net RCA, padlock probe RCA, target circle RCA and stride RCA. The paper also discussed the problems in the process of rolling circle amplification. We mainly introduced the application of RCA in basic research, the practical detection, medical diagnosis and Nano-materials. At last, the expectation towards the development of isothermal amplification of nucleic acid was prospected.

Application of SSR Markers in Variety Identification and Genetic Breeding of Vitis vinifera
DONG Zhi-gang1§, LIU Zheng-hai2§, LI Xiao-mei1,
2016, 6(2): 137-140. DOI: 10.3969/j.issn.2095-2341.2016.02.10
Abstract ( ) PDF (1041KB)( )

SSR(simple sequence repeat) has been widely used in the genetic breeding and germplasm resources research in recent years, because it has the characters of abundant quantity, reproducibility, high polymorphic, codominant inheritance and so on. The paper reviewed the application of SSR molecular markers in grape genetic diversity, variety identification, DNA fingerprint construction and genetic breeding. The application prospect of SSR markers in grape breeding was also presented.

Progress on Artificial Breeding and Application of Harmonia axyridis
SUN Bei-bei, YIN Zhe*, HOU Zheng-rong, LI Jin-
2016, 6(2): 141-145. DOI: 10.3969/j.issn.2095-2341.2016.02.11
Abstract ( ) PDF (1071KB)( )

Harmonia axyridis is a predatory natural enemy of aphids, scale insects, whitefly, spider mites and other agriculture and forestry pests. It has the characteristics of strong adaptability, high fecundity, large predating quantity, wide feeding habits and so on. The domestic and international development of artificial breading and application situation were reviewed in this paper to provide references for large-scale promotion and application of Harmonia axyridis.

The Comparation of Propagation Ability Between two Mouse Populations in Farm Fields of Shunyi District in Beijing
YUAN Zhi-qiang1, DONG Jie1*, QIAO Yan1, JIA Hai-
2016, 6(2): 146-150. DOI: 10.3969/j.issn.2095-2341.2016.02.12
Abstract ( ) PDF (2166KB)( )

In order to generate scientifically strategy on deratization, the paper analyzed the difference of population fecundity and its effect on population quantity between the two big species group of mouse, Apodemus agrarius and Cricetulus triton, in farm fields of Shunyi District in Beijing. During the years of 1994-2014, we investigated the mouse using clamping method during March to November (or January to December), settle 500 mouse traps in the monitoring points in the first third of each month. Specimen’s weights, body lengh, tail length, auricular height and hind leg length were measured, and their bodys were dissected to observe the progation. We analyzed the differences in fecundity between Apodemus agrarius and Cricetulus triton, results showed that Apodemus agrarius had more advantage in competition. Both Apodemus agrarius and Cricetulus had two propagation peak times each year and the main body of the propagulum were adult mice. Their offspring occupied 94.5% and 95.6% among all the infant mice, respectively. Both the propagation and main propagation of Apodemus agrarius were one month longer than those of Cricetulus triton. During the main propagation period, the proportion of male and female adult mice was higher than that of Cricetulus triton. The number of production was 1.7 times as many as that of Cricetulus triton. The average number of fetal mice was less than that of Cricetulus triton. These data indicated that the propagation ability of Apodemus agrarius were stronger than that of Cricetulus triton and had more advantage in competition.

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