| Glyphosate is a nonselective herbicide, it is mainly used to control perennial weeds and the non farmland weeds. With the continuous increase using of glyphosate, glyphosate resistant weeds were increasing in the world. Resistant Solanum nigrum L. were found in the field after using of glyphosate for a long time. Solanum nigrum L. is one of the main weeds in soybean fields. It caused the serious influence of soybeans’ growth, production and quality. In this paper, two kinds of seeds suspected resistant biotypes in the test field and one kind of seeds which was never treated with glyphosate in wasteland were gained. After sowing, Solanum nigrum L. seedlings which grew consitently were selected and treated them with 5 dose gradients to study physiology reaction on glyosate from different sources of Solanum nigrum L.. The resistance mechanism of glyphosate on Solanum nigrum L. was acquired through determination of the EPSPS gene sequences and protein expression with rapid amplification of cDNA ends in Solanum nigrum L.. The main results were as follows:(1) The study of resistance degree on glyphosate from different sources of Solanum nigrum L. in greenhouse:The two kinds of seeds suspected resistant biotypes in the test field and the one kind of seeds which was never treated with glyphosate in wasteland were named SA, SB and SC, respectively, and sowed them in the greenhouse. Glyphosate spray concentration were 0, 0.5, 1.0, 2.0, 4.0 kg ae/hm2, respectively.The ED50 of SB biotype is 0.8973 kg ae/hm2, ED50 of SC biotype is 0.7784 kg ae/hm2. And ED50 of SA biotype is 3.6878 kg ae/hm2. SA is 4.11 times more than SB, and 4.74 times more than SC.The accumulation of shikimic acid in the three kinds of biotypes were similar in 24-72 h after treatment with glyphosate, but there were significant differences in 72-216 h after treatment with glyphosate at 2.0 kg ae/hm2. The accumulation of shikimic acid in SA biotype declined shaply to the initial level in 120-216 h after treatment, but SB and SC biotypes sustained increased.(2) The study of resistant physiological reaction on glyphosate from different sources of Solanum nigrum L.:Glyphosate spray concentration were 0, 0.5, 1.0, 2.0, 4.0 kg ae/hm2 in this study, SC accumulated more shikimic acid than SA biotype. SC was 4.8 times more than SA biotype on the accumulation of shikimic acid after treatment with glyphosate at 2.0 kg ae/hm2. And with the increase of concentration of glyphosate application, the accumulation of shikimic acid increased.After application glyphosate at 2.0 kg ae/hm2 and 4.0 kg ae/hm2 on SA and SC for 3 days and 7 days, the fresh weight and dry weight were restrained on different extent, and with the concentration of glyphosate increased, the inhibition were enhanced. There was a significant difference between SA and SC biotypes at 7 days(P < 0.05), and the inhibition extent of SC biotypes was higher than SA.The chlorophyll content in both of the two biotypes changed after the application of glyphosate. 7 days after treatment with glyphosate at 2.0 kg ae/hm2, there was no significant difference between the SA biotype and the contrast, but the chlorophyll content in SC biotype was significantly reduced than the contrast. And the chlorophyll content of SA was 1.61 times than SC, which said that chlorophyll content in SC was significantly affected by glyphosate, and there was a significant difference between SA and SC biotypes on chlorophyll content after treated with glyphosate. At the same time, the two biotypes’ photosynthetic rate had also changed significantly. 7 days after treatment with glyphosate at 2.0 kg ae/hm2, there was no significant difference between the SA biotype and the contrast, but there was a significant difference between SC biotype and the contrast. And the photosynthetic rate of SA was 1.5 times of SC, which indicated that the photosynthetic rate of sensitive SC biotype was inhibited significantly by glyphosate.(3) The study of resistance molecular mechanism on glyphosate from different sources of Solanum nigrum L.:The cDNA length in SA- EPSPS gene is 1518 bp, encoding 505 amino acids, and the cDNA length in SC- EPSPS gene is 1539 bp, encoding 512 amino acids. Theoretical molecular weight were 124221.6 Da and 125932.4 Da, respectively. The fat index of SA was 27.47, and the instability coefficient of SA was 42.91 which was more than 40. The fat index of SC was 27.42 and the instability coefficient of SC was 42.98 which was also more than 40. Both of the two biotypes’ protein were belonged to unstable protein, and the theory of isoelectric point were both 5.01. SA- EPSPS and SC- EPSPS gene sequence consistency was 98.24%.The total number of amino acids and the species were different. The 25 th amino acid of SC-EPSPS was Asparagic acid, but the 25 th amino acid of SA-EPSPS was Asparagine, the base changed from G to A. The 109 th amino acid of SC-EPSPS was Valine, but the 109 th amino acid of SA-EPSPS was Aminopropionic acid, the base changed from T to C. The 157 th amino acid of SC-EPSPS was Ser, but the 157 th amino acid of SA-EPSPS was Phenylalanine, the base changed from C to T. There are three different amino acids between them. |
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