Table of Content

01 November 2014, Volume 47 Issue 21

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EFFICIENT, SAFE AND LARGE-SCALE TRANSGENIC TECHNOLOGY: OPPORTUNITIES AND CHALLENGES

Efficient, Safe and Large-Scale Transgenic Technology: Opportunities and Challenges

WAN Jan-min, LI Yu

Scientia Agricultura Sinica. 2014, 47(21):  4139-4140.  doi:10.3864/j.issn.0578-1752.2014.21.001

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Establishment and Application of Large-Scale Transformation Systems for Rice

ZHANG Xin, FU Ya-ping, ZHOU Jun-li, GUO Xiu-ping, LIU Wen-zhen, WU Jie-fang, WU Chuan-yin, WAN Jian-min

Scientia Agricultura Sinica. 2014, 47(21):  4141-4154.  doi:10.3864/j.issn.0578-1752.2014.21.002

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Rice, one of the most important food crops and a model monocot plant, its genetic transformation has attracted widespread attention. Since the world’s first case of transgenic rice got success in 1988, the rice genetic transformation technology has been developed rapidly. In 1994, in a milestone work of Agrobacterium-mediated transformation of a number of japonica rice cultivars, rice genetic transformation system has been more advanced after nearly 20 years of development. Presently, Agrobacterium-mediated transformation and microparticle bombardment are the two most widely used methods for rice genetic transformation, while pollen tube pathway, electroporation and polyethylene glycol (PEG) medium are also employed in some laboratories. Agrobacterium transformation is the method of choice because it is of easy and low cost, the high transformation efficiency, the low copy number of transgenes inserted into the host genome and the stability of expression over generations. Nowadays more than 80% of the transgenic rice is transformed by the Agrobacterium method. Although there are a lot of reports in improving the methods and technology of rice transformation, difficulties and limitations still exist in rice genetic transformation. The transformation efficiency of several elite japonica and many indica varieties is still unsatisfactory as it is much lower. Some transformation protocols are laborious, time consuming, and highly genotype-dependent. Besides, there is a problem of somaclonal variations or somatic mutation frequently occurs in plant cells during in vitro culture. Therefore, it is very important to establish the high efficient, safety, large-scale and standard transformation systems for diverse varieties of rice. This paper briefly reviewed the history and development of rice genetic transformation system. Especially, the recent progress of rice transgenic technology for large scale performance in China was described. Many factors are known to affect the efficiency of genetic transformation, such as the genotype of the recipient plants, the type and age of the tissue being inoculated, the strain of Agrobacterium, the expression vector, the composition of the culture medium, as well as various conditions of tissue culture. Extensive research has been conducted to optimize the high-efficiency transformation system for different genotypes of rice. Various factors affecting the transformation efficiencies were explored. Modification in culture conditions for embryogenic calli induction and regeneration from mature seed and also the compatibility of the Agrobacterium strain were successful in examined transformation protocols. The highly efficient Agrobacterium-mediated transformation system for japonica and indica rice using immature embryos or mature embryo-derived calli as the explant has been established for the large-scale production of transgenic rice by integrating new technology method. Also the Agrobacterium-mediated co-transformation system has been preliminary developed and some experimental parameters for marker-free rice transformation have been optimized. However, a big gap between the update status in China and the advanced level in some renowned international biotechnology companies is still existed in either transformation efficiency or transformation scale of rice. It was concluded that bio-safety, high efficiency and large scale were the bottlenecks for production GM rice products and its industrialization. Future studies in this field should be focused on the use of commercial rice varieties, improvement of transformation efficiency, development of marker-free rice plants, introduction of multiple target genes, integration of foreign genes at specific site, etc. The review also analyzed the problems existing in large-scale rice transformation system and provided strategies to improve it. It may provide some useful information for efficient production of transgenic rice for practical use as well as for studying gene function.

Establishment and Application of Large-Scale Transformation Systems in Wheat

YE Xing-guo, XU Hui-jun, DU Li-pu, HE Guang-yuan, WANG Ke, LIN Zhi-shan

Scientia Agricultura Sinica. 2014, 47(21):  4155-4171.  doi:10.3864/j.issn.0578-1752.2014.21.003

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Wheat is a plant that is relatively difficult to be genetically modified through traditional transgenic approaches among several major field crops. Factors restricting the transformation of wheat include small scale and low efficiency of transformation, poor repeatability, and fewer availability of transgenic materials with satisfied appearances, and thus leading to the lagged-behind of wheat in transgenic breeding compared to soybean, maize, cotton, and rice. Presently, transgenic techniques mainly used in wheat transformation are biolistic particle and Agrobacterium-mediated transformation based on in vitro tissue culture, although methods of pollen tube pathway, ion beam implantation, laser microbeam puncture, polyethylene glycol (PEG) medium, pollen-mediated, and Agrobacterium dipping floral have also been employed in some laboratories. In the aspect of recipient tissues, wheat immature embryo and its derived callus were mostly used as initiating materials for the transformation of target genes, but transgenic wheat plants were also reported to be obtained by using mature embryo, inflorescence, and anther derived callus, which still need to be investigated and confirmed. As for the transformation efficiency reported in wheat, 0.1%-16.7% is for particle bombardment and 0.7%-44.8% for Agrobacterium. A big variation still existed. In the matter of alien transferring genes for wheat, the target genes are mainly involved in wheat enhancement of flour quality, disease resistance, drought tolerance, aphids resistance, and herbicide tolerance except some selection or reporter genes such as nptⅡ, bar, hpt, GUS, GOX, pmi, and ALS. The transformation efficiency of wheat immature embryos mediated by Agrobacterium and particle gun is dramatically affected not only by genotypes, but also by the physiological status of mother plants which is determined by temperature, light, nutrition, and water supply during the growth period. Especially, the suitable day/night temperature during the period between anthesis and sampling of the donor plants is mostly beneficial to the embryonic callus induction and putative transgenic regeneration after transformation. Generally, the development of wheat transgenic technology in China has been largely progressed, for examples, the establishment of high regeneration system of wheat mature embryos and its successful application, optimization of regeneration and transformation system of wheat immature embryos, introduction of a group of interested genes related to wheat improvement in disease control, drought resistance and quality enhancement, preliminary setting up of wheat transformation system with larger scale, etc. However, there is still a big gap between the current status in China and the top-level international biotechnology companies regarding to the efficiency or scale in wheat transformation. It is thought that low transformation rate, strong genotype-dependence, and imperfect artificial climate conditions, as well as unstable manipulating team are the bottleneck to limit the application of wheat transformation technology by large scale in China. Future studies in this field should include the use of commercial wheat varieties, improvement of transformation efficiency, development of marker-free wheat plants, introduction of multiple target genes, integration of foreign genes at specific sites, avoidance of backbone insertion and transgene silencing, etc. Wheat transformation efficiency and scale will be improved and expanded by further modifications of plant regeneration system, exploration of regeneration and transformation-associated genes. Other aspects involve the screening of ideal genotypes, medium and technique improvement of co-culture, optimization and combination of various factors in transformation process. Development and application of biolistic particle and Agrobacterium mediated wheat transformation was reviewed in this article. Particularly, the progress of wheat transgenic technology for large scale performance in the latest five years was studied, which may provide useful information for the transformation of candidate genes for functional analysis and the breeding of transgenic wheat varieties.

Establishment and Application of Large-Scale Transformation Systems for Maize

LIU Yun-jun, JIA Zhi-wei, LIU Yan, ZHANG Deng-feng, LI Yu

Scientia Agricultura Sinica. 2014, 47(21):  4172-4182.  doi:10.3864/j.issn.0578-1752.2014.21.004

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Maize has the most widely planted scope and the largest output in the world, and makes important contributions to food security. However, pests, weeds, drought, salinity and other biotic and abiotic stress seriously affect maize production. The application of transgenic maize varieties with insect-resistance, herbicide-tolerance, disease-resistance and other traits could reduce the maize yield losses. Maize large scale transformation system has developed rapidly, and the transgenic insect-resistant and herbicide-tolerant maize varieties have also been commercially applied for 18 years, bringing huge economic, social and ecological benefits. Global biotech crop acreage increased from 1.7 million hectares in 1996 to 175 million hectares in 2013. The commercially used transgenic maize events were selected from a large number of independent transgenic events, and had several good characteristics, i.e. single copy of foreign genes, genetic stability, no vector backbone insertion, no effect on maize own traits. Foreign big companies and some public research institutions have established high efficient maize transformation system. In China, the study on maize transformation system started late, and currently the maize transformation system has been preliminarily established. However, it is necessary to further improve the efficiency and the scale of maize transformation system in China. Particle bombardment and Agrobacterium-mediated method are two main methods for maize transformation system. The article describes the principle, the development process and the application of these two methods. Agrobacterium-mediated transformation is the most mainstream of plant genetic transformation methods with the characteristics of low cost, low foreign gene copy number and stable gene expression, so Agrobacterium-mediated transformation method is more suitable for large scale maize transformation system. Over the last decade in China, some achievements have been made in maize transformation method by selecting good explants, optimizing Agrobacterium infection method. The established maize transformation system has also been used to develop a lot of valuable transgenic maize events with insect-resistance or herbicide-tolerance. The perspectives of the development of maize transformation were also discussed. The authors believe that the development trend of maize transformation system include: 1) screening explants from commercial maize inbred lines, 2) raising single-copy transformation event rates without enhancing vector backbone insertion, 3) multi-gene transformation technology, 4) gene targeting technology, 5) safety transformation technology. China should closely follow the development trend of maize transformation system, focus on the development of multi-gene transformation technology, gene targeting technology and safety transformation technology, to better serve the gene function research and the development of transgenic maize products. In China, if the transgenic maize events which were obtained by the large scale maize transformation system are commercially grown in the future, it will bring enormous economic, social and environmental benefits, and will promote the further development of maize transformation system. This review article might provide some useful information for the research on maize transformation system.

Establishment and Application of Efficient Transformation System for Cotton

LIU Chuan-liang, TIAN Rui-ping, KONG De-pei, LI Feng-lian, SHANG Hai-hong, CHEN Xiu-jun

Scientia Agricultura Sinica. 2014, 47(21):  4183-4197.  doi:10.3864/j.issn.0578-1752.2014.21.005

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This assay is a summary of the application of transgenic technology in cotton all over the world, including the establishment and research progress of cotton transgenic technology for large-scale performance in China in recent years, the main problems and development trend of transgenic technology in cotton, which gives an insight into the transgenic cotton for researchers and makes a great significance to help them working efficiently. Transgenic technology has achieved great progress in cotton bollworm resistance by GMOs, and will gradually get important progresses in disease and adverse resistance in cotton, etc. Early transformation events are transformed by Coker cultivars, and nowadays rapid progresses have been made in transgenic technology for the success of different cotton species of tissue culture in Gossypium raimondii, G. arboretum, G. barbadense, G. hirsutum, etc. and three major transformation methods containing biolistic particle, pollen tube pathway and Agrobacterium-mediated transformation. Cotton transgenic technology for large-scale performance in China is established mainly by Cotton Research Institute of Chinese Academy of Agricultural Sciences and other institutes in China. It forms a trinity system for cotton large-scale transformation by focusing on the optimization of Agrobacterium-mediated method through the selection of efficient transformation vectors, transformation system establishment of major cotton varieties or lines, optimization of tissue culture conditions, in addition to the improvement of biolistic particle and pollen tube pathway methods. This transformation system involves in establishment of Agrobacterium mediated transformation system selecting CRI24 as the transgenic receptor, getting new materials such as W12 whose differentiation rate is up to 100% by petiole tissue culture screening and transformation efficiency increased by 2.88 times compared with before, development of embryonic callus transformation system by biolistic particle method, improvement of the transformation efficiency by pollen tube pathway. Using the transform system, many transgenic materials or lines are obtained and some of them are bred to insect-resistant cotton varieties, more than 200 genes for 41 scientific research institutes in China have been identified their gene functions, and provides a large number of new materials for cotton breeding. It is concluded that strong genotype-dependence is the bottleneck for cotton transformation, expanding the genotype of transgenic receptor, improving the transformation efficiency, and scaling up the system are the subjects of cotton transformation for a long time. At the same time, concerning the efficiency and the public anxiety, further studies in this field should be focused on exploring and discovering safer and more effective transformation system such as multi-gene transformation, plastid transformation, fixed-point conversion or gene stacking, developing safe or maker-free transgenic technique. At last, the strict requirement for safety evaluation needs to be further researched on the genetically modified materials. With the publication of cotton genome sequences, it will be a new direction for cotton basic and applied research to clone cotton genes, which will provide valuable information for the transformation of candidate genes and breeding of transgenic cotton varieties.

Establishment and Prospect of Efficient Transformation Systems for Soybean

HOU Wen-sheng, LIN Kang-xue, CHEN Pu, JIA Zhi-wei, ZHOU Yang, YU Yang, LIU Yan-hua

Scientia Agricultura Sinica. 2014, 47(21):  4198-4210.  doi:10.3864/j.issn.0578-1752.2014.21.006

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Soybean [Glycine max (L.) Merr.] is the genetically modified (GM) crops that is earliest commercially planted on large scale. GM soybean is retaining its position as the GM crop occupied the largest scale globally with food, nutritional, industrial, and pharmaceutical uses. From 1996 to 2012, total 763.1 million hectares GM soybean had been planted and by growing GM soybean, almost 37 billion US dollars income was brought to farmers. In 2013, about 84.5 million hectares of GM soybean were planted around the world in 11 countries which accounted for 48% of all the GM crops hectarage and 79% for soybean grown all over the world. Though herbicide-resistant transgenic soybean holds a leading market share in the world, soybean has been regarded as a recalcitrant crop to be transformed for many years. Efficient and stable transformation is a restrictive factor for production of transgenic soybean and gene function research. The most widely and routinely used transformation systems are cotyledonary node-Agrobacterium-mediated transformation and somatic embryo-particle-bombardment-mediated transformation. The first fertile transgenic soybean plants were produced nearly simultaneously by these two methods in 1988. Even after more than two decades, these two methods have continued to be improved and have produced most transgenic soybean plants. The transformation efficiencies of these improved protocols have been extended and the system was successfully adapted to embryogenic suspension cultures for the regeneration of fertile transgenic soybean plants. These ready systems enable us to improve agronomic characteristics or to analyze gene function in soybean by transgenic approaches. In this review, recent advances and problems in research of soybean transformation system were described, with a focus on the characteristics of soybean regeneration systems such as organogenesis, somatic embryogenesis and protoplasts system. The representative soybean transformation systems mediated by Agrobacterium tumefaciens and particle bombardment have been summarized, and described their experimental parameters such as soybean genotype, explant, regeneration system, screening system, transformation frequencies. Analysis suggests that the genotype, explant type and so on can be used in the transformation of soybean has been developed and the transformation frequencies has been improved significantly. Some reports showed higher transformation efficiency over 10% and even some over 30% in some cases. However, the date of transformation frequencies had been obtained in some research reports that involved small sample size. The transformation frequencies between different experimental replications often repeated differences greatly. Sometimes, obtained high transformation frequencies in the single factor experiment, often doesn’t get well again in multi-factor integrated experiment. It indicated that the transformation efficiency still is affected by some unknown factors that is hard to control. On the other hand, the transformation efficiency still relies on the skill of the technicians and on the tissue culture conditions of the laboratory. That leads to some problems such as the same transformation process is poor in reproducibility between laboratories, the transformation frequencies are greatly different between operators, the transformation frequencies are not stable in the same laboratory. And those problems hindered the establishment of high-throughput transformation systems for soybean. Meanwhile, the research result of soybean transformation and transgenic lines was reviewed, and the current transgenic lines for soybean breeding in China were surveyed. Further, some new genetically modified technologies used in soybean were prospected, such as genome editing techniques and precise genome modification techniques.

Construction and Application of a Scale Transgenic Technology System for Pigs

MU Yu-lian, RUAN Jin-xue, WU Tian-wen, CHENG Ying, WEI Jing-liang, FAN Jun-hua, LI Kui

Scientia Agricultura Sinica. 2014, 47(21):  4211-4223.  doi:10.3864/j.issn.0578-1752.2014.21.007

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Swine is an important economic animal and pig breeding plays an important role in the development of agriculture in China. At the same time, as a kind of commonly used experimental animals, pigs are similar in anatomy, physiology and genetics to humans and important for life science research. To meet the specific needs of human beings, animal genomes can be modified by transgenic technology using genetic engineering or other experimental techniques, and the target gene can be passed on to the offspring steadily. Therefore, establishment of a scale swine transgenic technology system is essential for animal breeding, gene function and human disease model research. This paper reviewed the research progress of transgenic swine technology system both in China and foreign countries, summarized the principle of the selection of target genes, described the relative merits and applicable scope of specific gene transfer method such as pronuclear microinjection, somatic cell nuclear transfer, zinc-?nger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/ Cas9), sperm vector and lentiviral vector and so on, expounded the relevant regulatory element and system of gene expression regulation strategy. In recent years, China has made great progress in scale transgenic technology system research since the commencement of National Transgenic Major Projects: including explored a large number of functional genes; built ZFN, TALEN, CRISPR/Cas9 mediated genome modification technology platform; optimized the isolation, culture and screening process of donor cells; improved the somatic cell nuclear transfer and pronuclear microinjection system, and combined them with the novel genome editing techniques for site-specific, safe and efficient integration of exogenous gene; through a further cloning, embryo transfer, artificial insemination and other conventional breeding techniques integrated innovation, finally established the transgenic pig propagation technology system, so that the number of transgenic pigs can be expanded in a short time; provided technical service and trained technical staff for many domestic institutions, made transgenic technology to get a wider range of applications. But a series of problems still exist in the scale swine transgenic technology system of China: the lack of domain genes controlling the transgenic pigs related traits and with independent intellectual property; the transgenic technology supporting system is still not perfect; the potential risks of marker genes; the low integration efficiency and instability of exogenous gene; the inefficiency of gene targeting technology; the low piglets survival rate and so on. All of these are considered to be the bottlenecks of development of scale swine transgenic technology. Therefore, in order to realize the technological breakthrough of scale swine transgenic system, the important functional genes and regulatory element which have independent intellectual property must be explored by related omics tools, construct more efficient, safe and multi-gene transgenic vectors to achieve coordinated and efficient expression of exogenous genes, and improve the related technology in the preparation process of transgenic pigs. In conclusion, the transgenic swine breeding of China will enter a new high-speed development period under the support of scale swine transgenic technology system.

Establishment and Prospect of Efficient Transgenic System for Cattle

MENG Qing-yong, LIU Chun-cheng, WANG Meng, ZHANG Kuo, DAI Yun-ping, GUO Ying, FEI Jing, LI Ning

Scientia Agricultura Sinica. 2014, 47(21):  4224-4233.  doi:10.3864/j.issn.0578-1752.2014.21.008

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With the continuous development of life science and the expanding needs of industry application, transgenic technology, a basic technology of life science research, is expanding to life science industry application, which includes big domestic animal transgenic technology industrialization application. GM technology is discussed in this paper about the application and the development in transgenic cattle breeding, combining with the history of transgenic cattle in the world and the bovine transgenic breeding system construction in China. Through introduction, integration and innovation of the modern animal biotechnology, a safe, efficient and large-scale cattle transgenic technology system was established, which should improve the implement in the production of cattle and more extensive application in breeding practice. The purpose of transgenic technology for cattle breeding is improvement of the ability of disease resistance, the milk quality, the milk or meat output and the bioreactor of producing some functional proteins. At present, the scale GM technology system platform of cattle is nearly mature, which level has attained world class performance. More in-depth work will be focused on the regulation of functional gene and security control of import methods. In the future, the bovine transgenic breeding work should be led to “safe, efficient, large-scale”, which includes tissue specific promoters, optimizing codon, adjusting the genetic modification, point knock-in genes, marker free and so on. Disease-resistant and high-yielding dairy breeding is the essential direction of the development of the livestock industry. Some active functional proteins could be produced by cattle gland reactor, so establishment of a perfective efficient transgenic system for cattle is one of the prominent development direction in the future.

Approaches of Establishment of Technological System of Transgenic Sheep

LIU Ming-jun, ZHANG Xue-mei, LI Wen-rong, HUANG Jun-cheng, WANG Li-qin

Scientia Agricultura Sinica. 2014, 47(21):  4234-4245.  doi:10.3864/j.issn.0578-1752.2014.21.009

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Transgenic approach can break-through the barrier of breeds, integrate multiple gene effects and modify the genome based on the requirement of mankind, therefore it exhibits great potentials. Since the first transgenic animal born in 1982, more than ten species of transgenic animals have been reported. The methods to generate transgenic animals were developed from original pronuclear microinjection to nuclear transfer, viral vector transgenesis, and concurrent genome editing. The aims of the transgenic animal are also expanded from establishment of transgenic animal technique to generation of transgenic model, improvement of the productive performance or product quality, as bioreactor to produce high value pharmaceutical proteins, and breeding new animal breeds. Hereby the worldwide approaches, the faced problems, and the tendency to produce transgenic sheep were summarized. The proceedings of generation of transgenic novel breeds or animal models, and the characteristics of transgenic techniques were reviewed. The barrier and bottleneck of transgenic techniques were also addressed. Particularly, the feature, tendency and potential of the concurrently highlighted genome editing technique were comprehensively documented. Moreover, the necessity to establish a technological system of transgenic sheep was elucidated, and the present status, gain of innovation and breakthrough, highlighted target of transgenic sheep in domestic in future were reviewed. Meanwhile, the outlook of the economic, ecological and social effects resulted from transgenic sheep were further analyzed and previewed as well.

TILLAGE & CULTIVATION·PHYSIOLOGY & ECOLOGY

Study on Spatial Differences of Late Frost Injury to Winter Wheat and Its Reasons at Field Scale

WU Yong-feng, HU Xin, ZHONG Xiu-li, Lü Guo-hua, REN De-chao, SONG Ji-qing

Scientia Agricultura Sinica. 2014, 47(21):  4246-4256.  doi:10.3864/j.issn.0578-1752.2014.21.010

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【Objective】At the field scale, the study was conducted to investigate spatial differences of late frost injury to winter wheat and their influences on yields. The reasons for the spatial differences were discussed, which might provide a priori knowledge for predicting late frost damage and regulating influence factors.【Method】A frost-prone farmland in Shangqiu was selected as the study area where a winter wheat cultivar Aikang58 was sowed. Natural frost occurred in April 7, 10 and 21, 2013, when the winter wheat was in the periods from middle jointing to booting stage. At maturity, ear number and actual yield per square meter, and soil fertilities were respectively measured based on one hundred sampling points (the interval between samples is 5 m). Four indexes, such as dead ear rate (DER), injured ear rate (IER), injured ear index (IEI) and yield loss rate (YLR), were established to evaluate late frost damage. Their spatial differences and relations with ear number and actual yield were estimated using multiple linear stepwise regression method, geo-statistics method, cluster method, ANOVA and multiple comparison methods. In order to reveal the reasons for the spatial differences, late frost indexes related to developmental progressions of winter wheat and soil fertility factors were measured at early returning green stage. 【Result】 Through the stepwise regression, DER was found to be the only factor affected ear number and showed a negative effect. Factors, including IEI, DER and IER, all showed negative effects on actual yield. Of all the factors, IEI was the most powerful (its direct path coefficient reached -0.453) to actual yield. DER, IER and IEI were the factors impacted YLR and all showed positive effects, indicating that YLR increased with their increments. DER showed the most influence (its direct path coefficient was up to 0.626) on YLR. Late frost damage had a positive spatial autocorrelation (P＜0.05). Sampling points with similar frost damage degrees tended to the clustering distribution in the local space. Of all the damage evaluation indexes, YLR showed the strongest clustering distribution (Moran’s I=0.5538). The result of frost damage divisions indicated that ear number and actual yield significantly decreased (P＜0.05) with deepening frost injuries. The index DER showed the biggest increase (the amplitude reached 271.3%), followed by IER (36.4%) and IEI (31.8%). They comprehensively led to a sharp rise of YLR (132.1%). Sampling points with the most severe frost injury almost showed a contiguous distribution. Developmental progression of winter wheat and soil nitrogen in the turn-green stage showed obviously spatial differences and had a certain spatial correlation with late frost damage. A significant difference (P＜0.05) was found between total nitrogen, hydrolysable nitrogen, available phosphorus, available potassium and organic matter and the frost evaluation indexes. As soil nutrient content reduced, frost damage had a deepening trend. After the green up, the persistent drought made the soil water content decreased rapidly, further exacerbating the extent of frost damage. 【Conclusion】 At the field scale, the spatial differences of ear number and actual yield influenced by late frost damage were obvious. The spatial pattern of frost damage was significantly related to the developmental progress of winter wheat and soil nitrogen at returning-green stage, indicating that it maybe has an opportunity to predict frost damage risks early and regulating influence factors in the fine scale.

A Risk Assessment System of the Main Meteorological Disasters for Maize in Northeast China

GAO Xiao-rong, WANG Chun-yi, ZHANG Ji-quan, WEN Xu

Scientia Agricultura Sinica. 2014, 47(21):  4257-4268.  doi:10.3864/j.issn.0578-1752.2014.21.011

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【Objective】 Agricultural production is often threatened by several meteorological disasters. Presently, the risk assessment of agro-meteorological disasters often focuses on the influence of single meteorological disaster on disaster-affected body, not exactly reflecting the comprehensive risks faced by agriculture under real meteorological conditions. Meteorological disasters occurred at different crop growth stages have different influences on the final crop yield. In this paper, maize in Northeast China is used as an example to study risk assessment method and technology aiming at meteorological disasters at different crop growth stages. 【Method】 The whole growth period of maize in Northeast China was divided into four growth stages, namely sowing to seven-leaf, seven-leaf to tasseling, tasseling to milky ripening and milky ripening to maturation, and chilling, drought and flood are regarded as the three main meteorological disasters. Then with data on maize development record, meteorology, soil, crop sown area and yield, etc., and in accordance with the theory of natural disaster risk, the formation mechanism of agro-meteorological disaster risk and the “Four Elements” causing natural disaster risk, namely the natural disaster risk in certain area is caused by four factors, including hazard of the natural disaster, exposure and vulnerability of the disaster-affected body and the disaster prevention and mitigation capacity of human beings, the four elements for Northeast China’s maize of each growth stage were comprehensively analyzed, and risk assessment indices were selected thereby. Multi-indexes method for environment causing the disaster was adopted to select indices on meteorology, crops, physical geography, etc. in order to fully reflect hazard of the main meteorological disasters of each growth stage; assessment indices were selected according to the connotation of exposure and vulnerability of disaster-affected body; based on the relative lag situation of disaster prevention and mitigation, variation coefficient of yield was used to comprehensively reveal the preventing and mitigating capability, and a fairly complete index system of the main meteorological disasters risk assessment was developed. Owing to multiple and complicated factors causing agro-meteorological disasters and the fuzziness and uncertainty of such indices, analytic hierarchy process (AHP) was used to judge the weights of “Four Elements” and the hazard factors for each growth stage. The main meteorological disasters risk assessment model for each growth stage was established using natural disasters risk index method. The main meteorological disasters hazard assessment model was built by adopting synthetic weighted mark method, and the weight coefficients were determined by the ratio of the frequency of the three main meteorological disasters in each growth stage, reflecting the relative seriousness of the main meteorological disasters. 【Result】Based on the risk assessment of the main meteorological disasters in each growth stage, the model of the main meteorological disasters risk assessment of the whole growth period was established using synthetic weighted mark method, and the weight coefficients of risk indices for each growth stage were determined according to correlation relationship between yield reduction rate and the main meteorological disasters risk indices. A fairly complete index system of the main meteorological disasters risk assessment based on the crop growth stages was established, the models of the main meteorological disasters risk assessment and of hazard assessment for each growth stage were developed, and the model of the main meteorological disasters risk assessment for the whole crop growth period was also constructed. Multiple agro-meteorological disaster risk assessment technology system based on Northeast China’s maize growth stages was established basically. 【Conclusion】The analysis of hazard indices and yield reduction rate in the key development stage of the research stations in the typical widespread disaster years and of the representative stations in the typical disastrous years in Northeast China showed that the main meteorological disasters hazard indices were well selected.

PLANT PROTECTION

Density-Dependent Effect of Liriomyza trifolii at Immature Stage and Interspecific Competition with Liriomyza sativae

YI Hao, WANG Kai-ge, ZHANG Lin-ya, LEI Zhong-ren, LUO Hong-wei, LIAN Zhen-min, ZHOU Guo-qi

Scientia Agricultura Sinica. 2014, 47(21):  4269-4279.  doi:10.3864/j.issn.0578-1752.2014.21.012

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【Objective】The objective of this study is to determine how the density of Liriomyza trifolii eggs and larvae affect its growth and development, explore its density effect of intraspecific competition. In addition, the competition ability for food between L. trifolii and L. sativae larvae under different densities of 1st instar larvae were preliminarily described, and the replacing mechanism about interspecific competition between the two leafminer species was explored.【Method】The extra eggs and larvae of leafminers which were raised in laboratory were killed with needle, so as to form the different egg and 1st instar larvae density gradients, then the remaining leafminers were raised. According to the method of judging the lethal factor of insect whether existed density dependence, N₁ was used as individual numbers before the lethal factor acting on, N₂ was used as individual numbers after the lethal factor acting on. Moreover, lgN₁ as a dependent variable and lgN₂ as an independent variable, then the regression coefficient was calculated, while lgN₁ as an independent variable, lgN₂ as a dependent variable, the regression coefficient was calculated again. If this two straight lines were located in the one side of bisectrix b=1, the factors acted on N₁→N₂ were density dependent factors. Whereas if this two straight lines were located in two sides of the bisectrix b=1, this factor was not considered as a density related factor. By selecting one pair, two pairs and three pairs of adults of L. trifolii and L. sativae, and they were put on the fixed blade for 24 hours for spawn, in order to form different egg and larvae densities. Then the growth status of two leafminer species offspring was estimated, and the effect of different egg and 1st instar larvae densities on interspecific competition replacement was also analyzed. 【Result】 There was no density effect in L. trifolii during its egg stage, and the survival, growth and development rates were not significantly affected by egg density. Moreover, the survival rate of L. trifolii was above 91% under laboratory conditions. However, there was an obvious density effect at larval stage, in particular, the larvae density had significantly impacts on the survival rate of larvae, average weight of pupae, pupation rate, eclosion rate, lifetime of adults and egg laying amount of single female. When the 1st instar larvae density was larger than 0.87/cm² on the host plants of dwarf bean, larvae mortality was increased, the average weight of pupae, pupation and eclosion rates were reduced, the lifetime of adults and reproductive ability were also reduced. While the density of 1st instar larvae had no effect on the development rate of larvae and pupae, the female proportion of eclosion adults. In addition, the density during egg stage had no effect on the competitiveness of the two kinds of leafminers, the general hatching rate of the two kinds of leafminers under different densities were all above 94.25%. The higher larvae density led to the drastic interspecific competition. When the density of the two insects was low, their offspring numbers were similar. While the offspring numbers of L. trifolii was remarkably larger than the L. sativae when the density was high. 【Conclusion】 The egg density of L. trifolii had no significant effect on their population amounts. However, the higher 1st instar larvae density led to strong intraspecific competition, while it would refrain the growth of L. trifolii population. Compared with L. sativae, the interspecific competition ability of L. trifolii was much stronger, because the L. trifolii larvae have variable abilities to resist crowd, adapt for the aggravated environment and occupy food resources.

Characteristics of Rice Leaf Surface and Droplets Deposition Behavior on Rice Leaf Surface with Different Inclination Angles

XU Guang-chun, GU Zhong-yan, XU De-jin, XU Xiao-long

Scientia Agricultura Sinica. 2014, 47(21):  4280-4290.  doi:10.3864/j.issn.0578-1752.2014.21.013

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【Objective】In order to provide a basis of mechanisms controlling retention of pesticide droplets on target leaf, characterization of rice leaf surface and behavior analysis of single droplets of trisiloxane surfactant (Silwet-408) solutions on rice leaf surface with different inclination angles were studied.【Method】Scanning electron microscope (SEM) was used for observation of rice leaf surface characteristics and the critical surface tension (CST) of rice leaf was determined by Zisman method. Surface tension of Silwet-408 solutions at concentrations of 0, 3.91, 7.81, 15.63, 31.25, 62.50, 125.00 and 250.00 mg·L^-1 was measured and the critical micelle concentration (CMC) of Silwet-408 was also measured according to the change of surface tension of Silwet-408 solutions. Then the contact angle of a single droplet on the rice leaf surface with 3 inclination angles was determined by contact angle meter.【Result】SEM images showed that 3 types of hairs and densely covered papillae were observed on both the adaxial and abaxial sides of rice leaf. Densities of papillae, with significant difference between the adaxial and abaxial rice leaf surface, were ((12.4±0.7)×10³) and ((7.6±0.8)×10³)/mm², respectively. In contrast, no significant differences in stomatal length or stomatal density were found. The CMC of Silwet-408 was 78.5 mg·L^-1 and surface tension value of correspondingsolution at CMC was 20.77 mN·m^-1. The estimated CST values of the adaxial and abaxial rice leaf surface were 29.90 and 31.22 mN·m^-1, respectively. Among the measured Silwet-408 solutions, the droplets of solutions at lower concentrations (0, 3.91, 7.81 mg·L^-1) rolled off rice leaf with different inclination angles on condition that their surface tensions were more than the CST of rice leaf and Silwet-408 concentrations were less than the CMC. Surface tensions of solutions at concentrations 15.63, 31.25, and 62.50 mg·L^-1 were less than the CST of rice leaf and Silwet-408 concentrations were less than the CMC. Droplets of solutions at concentrations 15.63 and 31.25 mg·L^-1 adhered to rice leaf with lower inclination angle (30°). On the contrary, droplets rolled off rice leaf with higher inclination angle (60°). Droplets of solutions at 62.50 mg·L^-1 adhered to rice leaf and significant differences existed in decreasing speed of θ variation and wetting hysteresis. The droplets of solutions at higher concentrations of 125.00 and 250.00 mg·L^-1 adhered to rice leaf with different inclination angles on condition that their surface tensions were less than the CST of rice leaf and Silwet-408 concentrations were more than the CMC. After 40 s, no significant differences were observed in decreasing speed of θ variation and wetting hysteresis. Analysis results of advancing and receding angles on rice leaf surface with different inclination angles showed that advancing angles (θ_a) were larger than receding angles (θ_r). Within 40 s, both θ_a and θ_r decreased gradually.【Conclusion】The higher hydrophobicity of rice leaf is mainly ascribed to densely covered wax papillae on rice leaf surface and it may be related to hair length and stomatal density of rice leaf. Rice leaf surface is low energy. When surface tensions of Silwet-408 solutions are less than the CST of rice leaf surface and the concentrations of Silwet-408 are more than the CMC, droplets would show better adhesion on rice leaf surface with different inclination angles and wetting. Because of larger surface tension, droplets of low concentration solutions on rice leaf surface with different inclination angles are easier to roll off. That θ_a of droplets of Silwet-408 solutions on rice leaf surface with different inclination angles are always larger than θ_r illustrates wetting hysteresis. The phenomenon of wetting hysteresis indicates that rice leaf surface is rough and roughness is closely related to e.densely covered papillae on rice leaf surfac

SOIL & FERTILIZER·WATER-SAVING IRRIGATION·AGROECOLOGY & ENVIRONMENT

Capacity and Characteristics of Mineral Associated Soil Organic Carbon Under Various Land Uses

CAI An-dong, XU Xiang-ru, ZHANG Xu-bo, XU Ming-gang, ZHANG Wen-ju

Scientia Agricultura Sinica. 2014, 47(21):  4291-4299.  doi:10.3864/j.issn.0578-1752.2014.21.014

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【Objective】 Mineral-associated organic carbon (MOC, ＜53 µm) is one of the most important stabilization mechanisms of soil organic carbon (SOC). The objectives were to explore the content, proportion, and variations of the mineral-associated organic carbon from China's major land uses of cropland, grass land, and forest, which have a great significance to SOC stabilization, soil carbon sequestration potential, and sustainable carbon management.【Method】Publications were searched by using Web of China Knowledge Resource Integrated Database, Wan Fang Database, Science Direct and Springer link with the three keywords “2000-2014”, “Soil organic carbon”, “China” and “Aggregate fractionation (＜53 µm)”. A data set was set up with 901 pairs of data from 111 published papers that reported soil organic carbon and fractionation. Four categories of land uses such as Cropland (n = 580), Grassland (n = 98), Forest (n = 193), and other (Orchards, Tea plantations, etc., n = 31) were included. Soils from cropland included six soil classifications: Black soil, Paddy soil, Brown soil, Fluvo-aquic soil, Red soil, and Gray desert soil. The variations analysis of the SOC content and MOC and the proportion in different land uses and soil classification were performed using Kruskal-Wallis H significance test. 【Result】 The median value of total SOC content and MOC were significantly different among the three land uses. The total SOC content in forest soil was 18.2 g·kg^-1, which was significantly higher than those in the soils from grassland and cropland (12.0 and 10.3 g·kg^-1, respectively). The content of MOC (12.0 g·kg^-1 soil) was also significantly higher than that from cropland and grass land (8.0 and 7.6 g·kg^-1 soil, respectively). There were significant positive correlations (P＜0.001) between total SOC and MOC in the three land uses. The proportion of MOC in cropland (74.8%) was significantly higher than those in grassland (67.8%) and forest (70.3%) soil. It was also found that the proportion of MOC varied significantly according to land use and soil classification. The MOC proportion of black soil was the highest and the median was 87.4%, followed by paddy soils (76.7%) and red soil (74.0%). However, the proportion of gray desert soil was the lowest (62.5%). Regression analysis showed that the proportion of MOC decreased significantly with the increase in total SOC (P＜0.05) in black soil, paddy and brown soils, which was relatively high in SOC content. The MOC content was significantly correlated with the fine soil particles (＜53 µm) in cropland, grassland and forest, indicating the MOC saturation degrees were 68.4%, 58.7% and 91.5%, according to Six (2002) estimation, respectively. 【Conclusion】 MOC is unsaturated and can be further enhanced both in cropland and grassland soils. Therefore, the fine soil particles (＜53 µm) from cropland and grassland still have a high carbon sequestration potential.

Effects of Nitrogen Levels on Characteristics of Dry Matter Accumulation and Source-Sink Activities of Radish

WANG Xue, ZHANG Kuo, SUN Zhi-mei, MIAO Ze-lan, LIU Huan, MA Wen-qi

Scientia Agricultura Sinica. 2014, 47(21):  4300-4308.  doi:10.3864/j.issn.0578-1752.2014.21.015

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【Objective】 The purpose of this study was to investigate the effects of different nitrogen levels on the characteristics of dry matter accumulation and source-sink activities of radish to provide a theoretical basis for the best technique of nitrogen management in radish production. 【Method】 A field experiment was conducted in Shangyi county of Zhangjiakou city, the main production area of radish in semiarid cold region of Hebei province with a variety of Chunlei suitable for this local area. Six nitrogen levels at 0 (N0), 84 (N1), 126 (N2), 168 (N3), 252 (N4) and 336 (N5) kg·hm^-2 were set to analyze the differences between the dry matter accumulation characteristics and source-sink activities of radish. 【Result】 Results indicated that, compared to the control, nitrogen fertilization significantly increased the radish yield by 35.69%-64.59%, and increased the dry matter accumulations of radish by 29.15%-94.85%, 10.73%-101.31%, 22.86%-33.57% and 30.33%-66.47% at the stages of seeding, rosette, root development and maturity, respectively. The yields and dry matter accumulation of radish leaves and fleshy roots all increased firstly and then decreased with the increase of nitrogen application rates. Results also showed that the optimal nitrogen fertilization lengthened the fleetly accumulation duration of dry matter of whole plants and fleshy roots, and enhanced the maximum accumulation rate. Meanwhile, the time of reaching maximum dry matter accumulation rate of fleshy root and the starting time of fleetly accumulation of total dry matter were also both advanced. Furthermore, the optimal nitrogen fertilization increased the sink activities and the dry matter accumulation rates of leaves at earlier stage, and enhanced the source activities and the dry matter accumulation rates of whole plants and fleshy roots during the whole growing period. 【Conclusion】 Optimal nitrogen supply effectively coordinated the characteristics of dry matter accumulation and source-sink relationship of radish, and ultimately enhanced the yield of fleshy root.

HORTICULTURE

Evolutionary Study of MAM Genes Which Function in the Process  of Glucosinolates Synthesis in Brassica rapa

LIU Jin, ZHANG Ji-fang, LIANG Jian-li, CHENG Feng, WU Jian, WANG Xiao-wu

Scientia Agricultura Sinica. 2014, 47(21):  4309-4317.  doi:10.3864/j.issn.0578-1752.2014.21.016

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【Objective】The objective of this study is to explore the duplication and evolution of Brassica rapa MAM which are key genes in the process of glucosinolates synthesis. This study will provide a theoretical basis for future MAM research. 【Method】 The synthenic relationship of MAM genes among Arabidopsis thaliana, Schrenkiella parvula and Brassica rapa were analyzed, according to B. rapa datebase BARD, conserved motif of MAM genes were predicted by online software MEME, the expression of MAM homologous genes in 93 materials of Brassica rapa was analyzed by software R, and the phylogenetic tree was constructed by software MEGA 5.05. 【Result】 The results showed that the MAM homologous genes were located in three subgenomes of B. rapa. Amongst seven MAM genes, five geneswhich are synthenic orthologs to A. thaliana mainly originated from whole genome triplication. The remaining two genes may derive from translocation. Motif analysis result indicated that there were many differences in both ends of protein sequences in BrMAM homologous genes, compared with AtMAM genes. Expression data showed that the expression level of these homologous genes was variable, Bra021947 and Bra018524 expressed little in several materials of B. rapa, the expression of Bra029356 could be detected in all materials. In the phylogenetic tree, BrMAM homologous genes and AtMAM genes were located in different branches, in addition to Bra018524. 【Conclusion】 These results indicated that the B. rapa MAM genes had an independent evolution after the divergence of B. rapa and A. thaliana.

Expression and Protein Interaction Analysis of Light Responsive bZIP Transcription Factor MdHY5

LI Hui-feng, WANG Xiao-fei, RAN Kun, HE Ping, WANG Hai-bo, LI Lin-guang

Scientia Agricultura Sinica. 2014, 47(21):  4318-4327.  doi:10.3864/j.issn.0578-1752.2014.21.017

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【Objective】Cloning of the key light responsive bZIP (Basic-leucine zipper) transcription factors from Malus domestica Borkh, gene expression and protein interaction analysis, which were determined to identify the function and molecular mechanism of MdHY5.【Method】First, semi-quantitative primers used for detecting expression levels were designed from the apple in order to identify the light-responsive bZIP genes. Differentially screened and identified a light responsive gene named as MdHY5 based on comparative analysis of homologous. Subsequently, the primers were designed to clone the MdHY5 gene. Phylogenetic relationship including the MdHY5 protein with Arabidopsis bZIP proteins was analyzed using MEGA5 and the multiple alignment of bZIP domain was constructed, while the conserved protein domain was predicted, spatio expression analysis was made to detect the MdHY5 in different organs. The expression of MdHY5 in response to various light qualities was detected by semi-quantitative RT-PCR based on promoter prediction analysis, combined with the Arabidopsis【Result】A bZIP transcription factor was isolated in apple through the light response analysis. Phylogenetic relationship showed that MdHY5 is a homolog of Arabidopsis AtHY5. This gene is located on chromosome 12 of apple genome, with the gene number MDP0000586302, MdHY5 gene contains four exons and three introns in genome structure, which is similar to its homolog. The length of the cDNA is 1 112 bp, with 214 bp in the 5′ untranslated region, 403 bp in 3′ untranslated region, and 495 bp of open reading frame, which encode a 164 amino acid residues. MdHY5 contains a leucine zipper structure typical (bZIP domain) in the C-terminal of the protein, with a nuclear localization signal domain (NLS domain) included. Expression analysis showed that MdHY5 is expressed in various tissues, with the highest level in leaves. Promoter analysis showed that multiple light responsive cis-acting element exists in the promoters of MdHY5, such as G-box, GT-1-box, I-box and as so on. Quantitative expression analysis showed that MdHY5 gene could be induced by white, blue and UV light. The prokaryotic expression vector was transformed into BL21 and the fusion protein presented mainly in the supernatant after sonication. Pull down assay showed that MdHY5 could interact with MdCOP1 protein in vitro. 【Conclusion】 MdHY5 is a light responsive bZIP transcription factor and homologous to AtHY5, which is the key transcription factor in photomorphogenesis of Arabidopsis, MdHY5 has similar genome structure and conserved protein domain to AtHY5, and can be interacted with MdCOP1. microarray database. Finally, MdHY5 gene was inserted into the prokaryotic vector pGEX-4T-1, and IPTG was used to induce the fusion protein expression in E.coli BL21. Then the fusion protein was purified and detected by Western blot. The interaction between MdHY5 and MdCOP1 was detected by pull down assay.

STORAGE·FRESH-KEEPING·PROCESSING

Advances in Study of the Effect of High Pressure on Quality and Microbes of Fresh-Cut Fruits and Vegetables

ZHANG Xue-jie, YE Zhi-hua

Scientia Agricultura Sinica. 2014, 47(21):  4328-4340.  doi:10.3864/j.issn.0578-1752.2014.21.018

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This article reviewed the research proceeding on fresh cut fruits and vegetables processed by high pressure technology, which involved in the effect of high pressure on sensory quality, nutritional quality, antioxidant capability, enzymes, microorganisms and cell structure of fresh cut fruits and vegetables. The future research direction was also discussed. High pressure processing (HPP) is a non-thermal food processing method that subjects foods (liquid or solid) to pressures between 50 and 1 000 MPa. The suitable HPP can improve the taste of FCFV products, while higher pressure (＞200 MPa) has an adverse effect on the sensory quality. The nutritional quality of FCFV products by HPP is affected by raw material and treatments, lower pressure can increase the antioxidant capability, compared to the adverse effect by the higher pressure. Most enzymes related to quality of FCFV products are steady at lower pressure and even some enzymes were activated at some pressures, enzyme activities are decreased significantly when pressure increased over 400 MPa and some enzymes are inactivated especially by ＞600 MPa. The degree of inactivation of micro-organisms depends on different factors: micro-organism type, amount of pressure, treatment temperature and time, etc., the inactivation of micro-organisms are observed at HPP, while there will exist a high risk if the micro-organisms are not inactivate completely by HPP, even FCFV products are stored at 4℃. The pressure ＜300 MPa can not inactivate micro-organisms in FCFV products completely, although it also depends on raw materials and the original populations of micro-organisms. HPP faced challenges against the control of safety of FCFV products, because higher pressure will lower the quality of FCFV products to be unacceptable, therefore, to find a new way like combined technologies instead of a single HPP will be an alternative solution. The pressure over 100 MPa shows an impact on cell structure, which dominates the changes of quality and microbe counts of FCFV products at HPP. To sum up, selecting appropriate varieties resistant to HPP, using more effective combined HPP technologies and adopting management of hazard analysis and critical control point, are the premise of HPP to be applied in FCFV industry, and also the effective measures to guarantee the quality and safety of FCFV products. For the future, the HPP researches of FCFV will face against the effect of HPP on cell structure by numerical analysis, flavor control at HPP, development of combined HPP technologies and evaluation of appropriate varieties resistant to HPP, etc.

RESEARCH NOTES

Cloning of PAL Gene in ‘Yali’ Pear and Its Expression During Fruit Development and Wounding

YAN Hong-bo, CHENG Yu-dou, HE Jin-gang, GE Wen-ya, YANG Kun, GUAN Jun-feng

Scientia Agricultura Sinica. 2014, 47(21):  4341-4348.  doi:10.3864/j.issn.0578-1752.2014.21.019

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【Objective】 The objective of this study is to investigate the phenylalanine ammonia-lyase gene (PAL) sequence characters and expression pattern during fruit development and tissue browning in ‘Yali’ pear.【Method】Got full coding domain sequence of PAL by homology-based cloning, the bioinformatic characteristics of the cloned PAL were analyzed using online service, PAL protein phylogenetic tree was constructed by MEGA 5.1 software, analyzed the expression pattern of different development stages of fruit and tissue browning of ‘Yali’ pear by real-time quantitative RT-PCR. 【Result】 Two PAL genes containing full length CDS were obtained and termed PbPAL1 and PbPAL2, respectively. The full length cDNA of PbPAL1 was 2 232 bp, containing 2 160 bp open reading frame, 60 bp 5′ untranslated region and 12 bp 3′ untranslated region (GeneBank Access Number: GU906268.1). The full length cDNA of PbPAL2 was 2 387 bp, containing 2 163 bp open reading frame, 14 bp 5′ untranslated region and 210 bp 3′ untranslated region (GeneBank Access Number: GU906269.1). Phylogenetic analysis indicated that PbPAL1 and PbPAL2 were classified into two different phylogenetic tree branches. PbPAL1 was highly homologous to PAL of Pyrus communis, while PbPAL2 was highly homologous to PAL of Prunus persica. Although the expression levels of the PbPAL1 and PbPAL2 were different in the peel, flesh and core, all of the expression patterns showed a down trend along with fruit development, and were significantly higher in core than those in peel and flesh at early development stage. Wounding treatment could rapidly induce browning of flesh, while slowly induce the browning of peel. In both flesh and peel, the expression levels of PbPAL1 and PbPAL2 were markedly up-regulated by wounding treatment. In peel, the expression level of PbPAL1 appeared a peak at 6 h after wounding treatment, while PbPAL2 mRNA level significantly increased at 48 h after treatment. In flesh, the expression level of PbPAL1 obviously increased at 1 h after wounding treatment, and PbPAL2 began to rise at 12 h after wounding treatment.【Conclusion】PbPAL1 and PbPAL2 belong to different PAL gene types, and their expression showed a decreasing trend along with fruit development, and up-regulated by wounding, thus, they were involved in tissue browning induced by wounding in ‘Yali’ pear.