Vector construction and verification
In order to verify the successful construction of K167 vector. First, it was verified by electrophoresis (Fig. 1A) and double enzyme digestion (Fig. 1B) to check the release of the insert. Subsequently, K167 was sent to Shanghai Bioengineering Co., Ltd. for sequencing. The sequencing results combined with the results of enzyme digestion proved that K167 vectors had been successfully constructed, and these vectors were used for subsequent Agrobacterium transformation.
PCR-based screening of transgenic rice plants
PCR was employed to validate the transgene insertion using SHP-F and SHP-R primers. The amplified PCR product of 750 bp band size validated the SHP positive transgenic rice plants. The strip gels of the appropriate size were recovered and sent to Shanghai Bioengineering Co., Ltd. for sequencing. The sequencing results proved that the gene fragments were correct, indicating that the genomes of these strains all carried foreign genes, and these 21 strains were identified as positive plants (Fig. 1D, E).
Southern blot analysis of transgenic rice plants
Southern blotting confirmed the site of transgene integration. To extract pure rice leaf DNA, repeated extractions using RNase A was performed to eliminate RNA contamination. Lane1 is the positive control, and this lane is where the target gene fragment is melted and combined with the probe, so this lane has only one band and the color is darker. Lane 9 is the negative control, and this lane has no band. K1, K2, K3, and K4 all show two bands in the lanes, so it can be determined that the number of copies of the target gene contained in the genomes of these four lines is double copy, which is a double copy line. K5, K6, and K7 all show a single band in the lanes, so it can be determined that the number of copies of the target gene contained in the genomes of these three lines is a single copy, which is a single copy line. Southern blot result showed that exogenous genes were integrated into the rice genome. (Fig. 2A).
Real-time Fluorescent Quantitative PCR(RT-qPCR)of transgenic rice plants
RT-qPCR validated the presence of the SHP gene in transgenic rice plants. GAPDH was used as an internal reference gene, and cDNA from the wild-type rice line was considered as a negative control. RT-qPCR detection was performed on the positive plants of transgenic rice, and the results proved that the exogenous gene has been transcribed and expressed normally in rice, and the expression level has certain differences in different periods. The SHP gene of transgenic rice was up-regulated during the flowering and maturity stages. The GLOX gene expression was normal during the flowering period and was significantly up-regulated during the maturity period (Fig. 2B, 2C).
Basta smear experiment
The Bar gene in the K167 vector serves as a screening marker, and it imparts resistance against Basta herbicide, also known as glufosinate. The Bar gene expression in transgenic rice was analyzed using Basta. The resistance of transgenic rice plants to Basta demonstrated the Bar gene expression (Fig. 2D). Conversely, negative control plants were found to be sensitive to Basta as they did not express the Bar gene.
Measurement of the agronomic traits
As shown in Figures 3.A, 3.B, 3.C and 3.D, the wild-type and transgenic rice both grow well and develop normally. There are no significant difference of the wild-type and K-series transgenic rice lines in grain morphology, panicle shape and whole plant morphology.
The agronomic data of the transgenic lines are shown in Table 2, and the agronomic data of the wild-type are shown in Table 3. In terms of plant height, the wild type is not significantly different from K8, K18, and K19 (P> 0.05), but is significantly different from other strains (P <0.05). In terms of ear length, wild-type and K1, K4, K7, K8, K9, K10, K12, K14, K15, K16, K18 were not significantly different (P>0.05), and were significantly different from other strains (P<0.05). In terms of the number of tillers, the wild type was not significantly different from K2, K3, K5, and K13 (P>0.05), but was significantly different from other strains (P<0.05). In terms of total grains per ear, wild type has no significant difference with K1, K4, K5, K7, K8, K16, K18, K20 (P>0.05), and significant difference from other strains (P<0.05). In terms of the number of grains per panicle, the wild type is not significantly different from K1, K4, K5, K7, K8, K11, K16, K20 (P>0.05), and significantly different from other strains (P <0.05). In terms of seed setting rate, there was no significant difference between wild-type and all transgenic rice (P>0.05). In terms of 1000-seed weight, wild type has significant differences with K2, K3, K6, K9, and K12 (P <0.05), but not significantly different from other strains (P> 0.05).
Measurement of peroxidase activity
Peroxidase activity was detected spectrophotometrically in transgenic plants, and these plants were identified through PCR-based screening using wild-type rice plants as control. Transgenic rice leaves demonstrated higher peroxidase activity than the control leaves, but activity levels varied notably among the transgenic rice leaves (Fig. 4A). According to SPSS analysis, the peroxidase activity of wild-type rice and transgenic rice lines are extremely different (P <0.01). It can be seen from Figure 4B that the peroxidase activity in the leaves of the transgenic rice at the mature stage is higher than that in the wild-type rice, and there are also certain differences between different transgenic lines. Compared with the wild type, the highest value of peroxidase activity of the transgenic rice line K4 increased by 65.38%, and the lowest value of K3 increased by 22.16% compared with the wild type. The results showed that the exogenous genes SHP and GLOX can be expressed normally and efficiently under the guidance of the promoter, the peroxidase activity of the transgenic rice plants was significantly increased.
Determination of heterocellulose content
Heterocellulose content is the sum of cellulose and hemicellulose content. However, when cellulose and hemicellulose content are measured separately, a partial loss in yield is witnessed, which results in an inaccurate measurement of lignin components. This inaccuracy can be eliminated by measuring the heterocellulose content. In this study, the holocellulose content of rice in the flowering stage is shown in Fig 4B. The holocellulose content of wild-type rice is 61.12% ± 0.89%, the highest holocellulose content in transgenic rice lines is K4, which is 62.85% ± 0.28%, and the lowest holocellulose content is K2, which is 60.48% ± 0.74%. Data analysis using SPSS 26.0 showed that the content of cellulose in wild-type rice lines was not significantly different from that of transgenic rice lines (P>0.05), indicating that the introduction of exogenous genes SHP and GLOX did not affect the synthesis of cellulose in plants during blooming. The total cellulose content of rice at the mature stage is shown in Fig 4C. The holocellulose content of wild-type rice is 63.35% ± 0.78%, the highest holocellulose content of transgenic rice lines is K6, which is 66.04% ± 0.87%, and the lowest holocellulose content is K15, which is 63.30% ± 0.52%. Data analysis using SPSS 26.0 showed that the content of cellulose in wild-type rice lines was not significantly different from that of transgenic rice lines (P>0.05), indicating that the expression of exogenous genes SHP and GLOX did not affect the synthesis of cellulose in plants at the mature stage.
Determination of lignin content
The lignin content of rice in the flowering stage is shown in Fig 4D. According to measurement, the lignin content of wild-type rice lines is 33.63% ± 0.76%, the highest lignin content of transgenic rice lines is K8, which is 35.90% ± 0.70%, and the lowest lignin content of transgenic rice lines is K10, which is 32.47. % ± 0.81%. Using SPSS 26.0 for data analysis, the lignin content of wild-type rice lines was not significantly different from that of transgenic rice lines (P> 0.05), and the lignin content of some transgenic rice lines was slightly higher than that of wild-type rice. Studies have shown that the exogenous gene SHP can promote the synthesis of lignin in the early stage of plant development, and can effectively degrade lignin under the action of H2O2 in the later stage. Therefore, it is speculated that the expression of the exogenous gene SHP does not affect the lignin synthesis in the early stage of the plant, and may also promote the lignin synthesis.
The lignin content of rice in the mature stage is shown in Fig 4E. According to the measurement, the lignin content of wild-type rice lines is 35.17% ± 0.84%. the highest lignin content of transgenic rice lines is K9, which is 30.45% ± 0.66%, and the lowest lignin content of transgenic rice lines is K5, which is 24.58. % ± 0.27%. Using SPSS 26.0 for data analysis, the lignin content of wild-type rice lines was significantly different from that of transgenic rice lines (P <0.01). It can be seen from Fig 5.E that the lignin content of transgenic rice at the maturity stage is lower than that of wild-type rice, and there are also certain differences between different transgenic lines. Compared with the wild type, the lignin content of the transgenic rice line, the highest value K9, was reduced by 13.42%, and the lowest value, K5, was reduced by 30.11% compared with the wild type. The results show that GLOX can provide H2O2 for SHP, and SHP can effectively degrade lignin under the action of H2O2. Therefore, it is speculated that the expression and interaction of exogenous genes SHP and GLOX can effectively reduce the lignin content.
Results of ethanol fermentation experiments of transgenic plants
Analysis of the Determination of Reducing Sugar Content
The reducing sugar concentration determined after cellulase hydrolysis is shown in Fig. 5A. The experimental data results represent three groups of duplicates. After the no pretreatment group and the treatment with ddH2O group, the contents of reducing sugars obtained from cellulase-degraded transgenic rice and the control (WT) were essentially same. However, after 50% sulfuric acid pretreatment, both the transgenic rice and the control showed an increase in the content of hydrolyzed sugars (23.2 g/L); this was significantly higher (p < 0.05) than that in the untreated group (5.8 g/L).
Determination of Ethanol Content
Ethanol fermentation experiments were performed on ddH2O treated transgenic rice, 50% sulfuric acid-pretreated rice, and non-pretreated rice. Compared with the same fermentation conditions, the three pre-fermentation treatments resulted in changes in the amount of ethanol produced. Under an initial fermentation pH of 5.0 and temperature of 30°C, and an inoculation amount of 10%, fermentation products were obtained at 24 h, 48 h, 72 h, and 96 h in order to determine the ethanol concentration. As shown in Fig 5B, the ethanol content determined at each period of pretreatment with 50% sulfuric acid was much higher than the corresponding value of the untreated group and ddH2O pretreatment. The final ethanol concentration of transgenic rice (9.1 g/L) was significantly higher than that of non-transgenic rice (2.6 g/L), but slightly lower than the concentration of the acid pretreatment group (9.9 g/L).