Li Jiu-di

Institute of Botany

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Yuemei Dong

Johns Hopkins University

Yongxing Li

State Key Laboratory of Information Engineering in Surveying Mapping and Remote Sensing

Zhu Zhi-qing

Anhui University of Finance and Economics

Qianli An

State Key Laboratory of Rice Biology

Lingang Lu

China People's Police University

Sui Wenzhi

Jin Hong-fan

Yan Wang

Shanghai Jiao Tong University

Jia Yuewei

魏

魏水建

Cooperative Institutions

Chinese Academy of Sciences

Chinese Academy of Medical Sciences & Peking Union Medical College

Sun Yat-sen University

Institute of Botany

Fudan University

China Agricultural University

East China University of Science and Technology

Capital Medical University

Institute of Chemistry

Institute of Botany

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Using confocal laser scanning microscope to visualize the infection of rice roots by GFP-labelled Klebsiella oxytoca SA2, an endophytic diazotroph

Zhiwu xuebao (2001)

Qianli An Xuejian Yang Yuemei Dong Lijie Feng Baijian Kuang

Microscopic observation showed significantly the process that the bacteria infected the roots from the root surface to the vascular tissues in the root maturation zone. After colonization on the root surface, the GFP-labelled bacteria penetrated preferentially into the cortex of the developed lateral roots and colonized inter- and intracellularly in the lateral root. Afterwards, they moved into the vascular cylinder at sites where the endodermis was modified by the outgrowth and the development of the lateral root. In addition, they also could penetrate into the cortex with less frequency and efficiency of the primary roots at sites where no lateral root was emerged. Cell walls of the root cells close to the invading bacteria were thickened and strong yellow-green fluorescence of them was visualized under blue light. The phenomena may represent a localized hypersensitive reaction of root cells to further restrict bacterial invasion.

Klebsiella oxytoca

Root hair

Endodermis

Root cap

Lateral root

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INOCULATION,ISOLATION OF ENDOPHYTIC DIAZTOROPHIC BACTERIA AND THEIR COLONIZATION IN MAIZE ROOT TISSUE UNDER GNOTOBIOTIC CONDITION

Chinese Journal of Appplied Environmental Biology (2001)

Li Jiu-di

This paper describes inoculation of 18 bacterial strains isolated from maize to maize, isolation, screening and identification of those strains based on morphology and color, using multi carbon medium, and REP PCR. The result indicated that two diazotrophic bacteria of R1 and R6 with high acetylene reduction activity were obtained. The plasmid of pKK223 GFP harboring gfp (reporter gene) was transferred into R1 and R6, respectively, and two transformants were obtained with the overexpression of GFP, designated as R1A and R6A; and pMGFP2.1 harboring nif H gfp was transferred into R1 and R6, and two transformants carrying pMGFP2.1 were designated as R1B and R6B, respectively. Also the research of colonizing R1A and R6A in maize root tissues, and inducing expression of nif H gfp in R1B and R6B was conducted gnotobiotically. The result showed that R1A and R6A were colonized in intercellular spacer, xylem vessels, wall of xylem vessels and parenchyma of stele predominantly. It was difficult for R1B and R6B to express nif H gfp in root tissues because of the limited nutrients and factors related to dinitrogenas gene expression. Only if 1‰ sucrose was supplemented, nif H gfp could be expressed. Fig 2, Tab 2, Ref 15

Parenchyma

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Survival regularization of enterobacter gergoviae-7 (E7) in different carriers and maize rhizosphere

Chinese Journal of Appplied Environmental Biology (2002)

Sui Wenzhi Lingang Lu Zhong Erong Tang Qing-fa Xie Ying Yan Wang

Peat, water and humic acid used as carriers were inoculated with the associated nitrogen-fixing engineered bacteriaEnterobacter gergoviae-7(E7).After the inoculants were stored at room temperature for 5 d, the E7 numbers in inoculants were increased obviously. After one month the E7 numbers in inoculants decreased slightly; and after two months fell rapidly. Six months later, the E7 numbers〔n(CFU)〕 in inoculants decreased to 5.0×10 6～93.0×10 6 g -1. The survival regularization of E7 in three inoculants was the same. E7 numbers in inoculants were peat water humic acid inoculants. After application of peat inoculants in maize seeds for 20 d, E7 numbers in the maize roots and maize rhizosphere were maximum. After inoculation for 20 d the E7 numbers decreased. After milking stage, no E7 was found in the maize roots and maize rhizosphere. The results of field plot inoculating experiment showed the highest yield of maize was obtained when the peat inoculants was mixed with seeds and at the same time sowed under 2 cm of seeds(Tab 3).

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Use of GFP and antibiotic resistance as selective markers to monitor colonization of associated nitrogen-fixing bacteria in maize rhizosphere.

Chinese Journal of Appplied Environmental Biology (2000)

Dong Yuemei Qianli An Li Jiu-di Zhu Zhi-qing

The chloramphenicol gene of plasmid pKRP10, the kanamycin gene of plasmid pKRP11 and tetracycline gene of plasmid pKRP12 were cloned into the Pst I site on the downstream of gfp gene of plasmid GFPmut2 respectively, the recombined plasmids had the gfp and different antibiotic resistance genes . These plasmids were transformed into Enterobacter gergoviae 57 7 and genetically engineered strain E7 respectively, the resulting transformants exhibited antibiotic resistance and appeared bright green under blue light. Based on the two kinds of selective markers above, the numbers of these transformants colonizing on maize roots were confirmed. The colonization of gfp labeled bacteria on the maize roots as well as their distribution in the soil was observed by fluorescent microscopy. Plate 1, Fig 1, Tab 2, Ref 15

Kanamycin

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The Construction of nifH-gfp Expression Vector and Its Expression in Enterobacter gergoviae 57-7

Wang Wei Yuemei Dong Qianli An Xianglin Liu Li Jiu-di

A nif H_ gfp expression vector pMGFP2 was constructed by fusing the 725 bp PCR amplified triple mutated green fluorescent protein ( GFP ) gene ( gfp S65T,V68L,S72A ) fragment to the nif H promoter and its start codon which was from Klebsiella pneumoniae (Schreter) Trevisan M5a1. A kanamycin cassette was inserted into Pst Ⅰ site of pMGFP2, obtaining the expressing vector pMGFP2.1 which can be used for the studying of nif H_ gfp expression in Enterobacter gergoviae 57_7. It was then transformed into E.gergoviae 57_7 and the effects of NH + 4 and oxygen on the expression of nif H_ gfp in E. gergoviae 57_7 were studied.

Kanamycin

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Characterization of Nitrogen Fixation in Maize Root Associated Klebsiella planticola 19-1

Journal of Integrative Plant Biology (1994)

Li Jiu-di Jiwen Wang Yongxing Li Yuemei Dong Jin Hong-fan

Klebsiella

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Isolation and characterization of NH4+-excreting mutants of Enterobacter gergoviae 57-7