Effect of heat treatment on the degradation of recombinant protein and recombinant DNA in transgenic straws

doi:10.3969/j.issn.1004-1524.20240332

Acta Agriculturae Zhejiangensis ›› 2024, Vol. 36 ›› Issue (9): 2079-2088.DOI: 10.3969/j.issn.1004-1524.20240332

• Environmental Science • Previous Articles Next Articles

Effect of heat treatment on the degradation of recombinant protein and recombinant DNA in transgenic straws

YAN Jingying(), NI Liang, SHEN Xingyu, LI Yu

Agricultural Experimental Station, Zhejiang University, Hangzhou 310058, China

Received:2024-04-11 Online:2024-09-25 Published:2024-09-30

Abstract

Abstract:

With the continuous expansion of transgenic crop planting area, how to efficiently deal with the transgenic straw has become an important scientific issue. The recombinant protein and recombinant DNA in untreated transgenic straw can persist in the soil for a long time and have a potentially negative impact on soil biodiversity. Therefore, it is of importance to find a cost-saving and environmentally friendly straw treatment method. High temperature treatment is an effective method to degrade the recombinant protein and recombinant DNA of transgenic straw, but the treatment temperature and treatment time have not been systematically studied at present. This study used the test strips and polymerase chain reaction (PCR) to detect the levels of recombinant protein and recombinant DNA in the transgenic straw under different temperature and time treatments, and found that phosphinothricin acetyltransferase (PAT) and other recombinant proteins in the straw of transgenic soybeans, cotton, maize and rice were almost degraded after being treated at 50 ℃ for 3 h. However, at the same temperature, the degradation of recombinant DNA took 4 d. The degradation time of recombinant proteins and recombinant DNA could be shorted by increasing the temperature. These combinations of temperature and time, particularly the treatment condition of 50 ℃ for 4 d, could be achieved by composting in agricultural production. Therefore, this study provided a theoretical basis for the treatment of transgenic straw from the perspective of molecular biology.

Key words: transgenic straw, recombinant protein, recombinant DNA, phosphinothricin acetyltransferase (PAT), CP4 5-enolpyruvyl-shikimate-3-phosphate synthase gene (Cp4-EPSPS), Bacillus thuringiensis (Bt) gene

CLC Number:

S141.4

YAN Jingying, NI Liang, SHEN Xingyu, LI Yu. Effect of heat treatment on the degradation of recombinant protein and recombinant DNA in transgenic straws[J]. Acta Agriculturae Zhejiangensis, 2024, 36(9): 2079-2088.

Figures/Tables 7

Table 1 Primer information

基因 Gene	正向引物序列 Positive primer sequence (5’→3’)	反向引物序列 Reverse primer sequence(5’→3’)	产物长度 Product length/bp
CaMV35S	GATAGTGGAAAAGGAAGGTGGC	GAAGGGTCTTGCGAAGGATAG	230
STOP1-GFP	CTCTGTTCCAGGGACACACG	GTCCATGCCGTGAGTGATCC	1 100

Fig.1 Effect of heat treatments with different temperature and time on the recombinant protein degradation of transgenic soybean straw A, Effects of different temperature and time treatments on PAT degradation of transgenic soybean straw; B, Effect of different temperature treatments on PAT degradation of transgenic soybean straw; C, Effect of different temperature treatments on Cp4-EPSPS degradation of transgenic soybean straw; D, Effect of different temperature treatments on Bt degradation of transgenic soybean straw. CK indicated treatment under normal temperature; N indicated negative control with detection of wild-type soybean straw. The blue arrow indicated quality control line, and the red arrow indicated detection line.

Fig.2 Effect of different heat treatments on GFP fusion protein stability in transgenic Arabidopsis A, GFP fusion protein in transgenic Arabidopsis root tip under normal temperature; B, C and D, GFP fusion protein in transgenic Arabidopsis root tip under different temperatures.

Fig.3 Effect of different heat treatments on GUS activity of transgenic Arabidopsis A, GUS staining of transgenic Arabidopsis leaf under normal temperature; B, C and D, GUS staining of transgenic Arabidopsis leaf under different temperatures.

Fig.4 Effects of 50 ℃ treatment for 3 h on the degradation of recombinant proteins in fresh straw of different transgenic crops A, PAT transgenic crop straw; B, Cp4-EPSPS transgenic crop straw; C, Bt transgenic crop straw. N indicated negative control with detection of wild type crops; CK indicated treatment under normal temperature; L indicated leaf; S indicated stem. The blue arrow indicated quality control line, and the red arrow indicated detection line. The same as below.

Fig.5 Effects of 50 ℃ treatment for 3 h on the degradation of recombinant proteins in mature straw of different transgenic crops

Fig.6 Effects of heat treatment at different temperatures and time on the stability of transgenic soybean straw DNA A, Effects of heat treatment at different temperatures and time on the stability of GFP fusion gene in transgenic soybean straw; B, Effects of heat treatment at different temperatures and time on the stability of CaMV35S sequence in transgenic soybean straw; The length of GFP fusion fragment was 1 100 bp, the length of CaMV35S fragment was 230 bp; M, DL 2 000 marker; Lanes 1-3 (N) indicated negative control of non-transgenic soybean straw, lanes 4-6 indicated transgenic soybean straw treated at normal temperature, lanes 7-9 and 16-18 indicated transgenic soybean straw treated at 50 ℃, lanes 10-12 and 19-21 indicated transgenic soybean straw treated at 70 ℃, lanes 13-15 and 22-24 indicated transgenic soybean straw treated at 90 ℃; * indicated non-specific band. C, Effects of different time of 50 ℃ treatment on the stability of CaMV35S sequence in transgenic soybean straw; Lanes 1-3 indicated fresh transgenic soybean straw, lanes 4-6 indicated transgenic soybean straw treated at normal temperature for 3 d, lanes 7-21 indicated transgenic soybean straw treated at 50 ℃ for different time, the samples in lanes 22-24 were ddH2O.

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Effect of heat treatment on the degradation of recombinant protein and recombinant DNA in transgenic straws

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