Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (6): 1104-1111.doi: 10.3864/j.issn.0578-1752.2021.06.003

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Effect of rhcN Gene Mutation on Nodulation Ability of Soybean Rhizobium HH103

HanXi LIU1(),Hao LÜ1,GuangYu GUO2,DongXu LIU1,Yan SHI1,ZhiJun SUN1,ZeXin ZHANG1,YanJiao ZHANG1,YingNan WEN1,JieQi WANG1,ChunYan LIU1,QingShan CHEN1(),DaWei XIN1(),JinHui WANG1()   

  1. 1College of Agriculture, Northeast Agricultural University, Harbin 150030
    2Harbin Academy of Agricultural Science, Harbin 150028
  • Received:2020-09-03 Accepted:2020-10-26 Online:2021-03-16 Published:2021-03-25
  • Contact: QingShan CHEN,DaWei XIN,JinHui WANG E-mail:396525797@qq.com;qshchen@126.com;dwxin@neau.edu.com;jinhuiwang113@126.com

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Abstract:

【Background】 Soybean is an important economic crop, which is the main source of food and feed. Symbiosis is a special character of soybean to fix nitrogen in air and transferred into ammonia via rhizobia. Via symbiosis soybean can acquire enough nitrogen source for development. Rhizobial type Ⅲ effectors play pivotal roles in regulating the establishment of symbiosis. The rhcN gene codes an ATP transferase, which can regulate the secretion of type Ⅲ effector of rhizobium into host cells. 【Objective】To elucidate the symbiotic mechanism will help to improve the nitrogen fixation efficiency of soybean-rhizobium symbiosis system and is useful for friendly agricultural development. 【Method】In this study, rhcN mutant was constructed by tri-parental mating method and inserted a kanamycin gene in the downstream of rhcN start codon. The mutant was confirmed by PCR and Southern blot. To detect whether the expression of other type Ⅲ effectors was affected by rhcN mutant. The expression of NopC and NopT were identified by induced by genistein via Western blot method. Nodulation experiment were performed in Leonard jars, to detect the nodule phenotype of Williams82 after inoculation with the wild strain HH103 and derived HH103ΩrhcN mutant. Finally, one-hundred soybean germplasms with different genotypes were used for nodulation experiment. 【Result】The results of PCR and Southern blot confirmed that HH103ΩrhcN mutant was successful, and the extracellular protein identification supported that rhcN mutant can inhibit the secretion of type Ⅲ effectors NopC and NopT. The nodulation test of Williams82 showed that rhcN mutation can significantly inhibit the nodule number and dry weight. Using HH103ΩrhcN mutant and wild type HH103, 100 core germplasm accessions were identified, these results showed that rhcN mutation could reduce the root nodule phenotype of 80 soybean accessions, significantly increase the root nodule phenotype of 13 soybean accessions, and 7 soybean resources did not change significantly. 【Conclusion】The results showed that the abnormal secretion of type Ⅲ effector factors of rhizobium could affect the establishment of symbiotic system. rhcN played an important role in the formation of soybean rhizobium symbiosis system, and different soybean genetic background had different responses.

Key words: soybean, rhizobium, type Ⅲ effector, rhcN, symbiosis

Fig. 1

PCR analysis of HH103ΩrhcN M: Trans 2K Plus DNA marker; 1: HH103ΩrhcN（rhcN-F,rhcN-R）; 2: HH103（rhcN-F,rhcN-R）; 3: HH103ΩrhcN（rhcN-F,Km-SpeⅠ-R）; 4: HH103（rhcN-F,Km-SpeⅠ-R）"

Fig. 2

Southern blot identification of HH103ΩrhcN 1: The genome of HH103ΩrhcN was digested by HindⅢ; 2: The genome of the wild strain HH103 was digested by HindⅢ; 3: The genome of HH103ΩrhcN was digested by SacⅠ; 4: The genome of the wild strain HH103 was digested by SacⅠ"

Fig. 3

Detection of NopC and NopT in secreted proteins +: The presence of Genistein; -: The absence of Genistein"

Fig. 4

Root morphology and nodules traits after HH103 and HH103ΩrhcN inoculation The left picture showed the information of soybean roots and nodules inoculated with HH103, and the right picture showed the information of soybean roots and nodules inoculated with HH103Ωrhcn"

Fig. 5

Nodule number and dry weight of HH103 and HH103ΩrhcN inoculation *for the difference was significant at P<0.05 level"

Table 1

Analysis of nodule traits in 100 germplasm accessions"

参数
Parameter		 HH103 HH103ΩrhcN
根瘤数目 NN 根瘤干重 NDW (mg) 根瘤数目 NN 根瘤干重 NDW (mg)
平均值 Average 35.52±5.64 34.37±6.35 17.04±10.56** 16.32±11.55**
最大值 Maximum 49.60±4.67 50.84±7.49 45.20±4.87 47.68±6.58
最小值 Minimum 11.50±4.84 12.98±3.65 3.00±0.87 0.35±0.26
标准差 Standard deviation 5.64 6.35 10.56 11.55
变异系数 Coefficient of Variation 54.24 61.02 101.57 111.07
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