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Hereditary and Phenotypic Analysis of Transgenic Tomato with Interleukin-4 Gene and Its F1 Hybrids

Author: PengHongYu
Tutor: YuLiJie
School: Harbin Normal University
Course: Botany
Keywords: tomato (Lycopersicon esculentum) interleukin-4 (IL-4) F1-hybrids genetic expression
CLC: Q943
Type: Master's thesis
Year: 2011
Downloads: 12
Quote: 0
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Abstract


The present paper focuses on the analysis of il-4 gene transgenic tomatoes in T5, T6 generation and F1-hybrids. The seed emergence ratio was recorded, PPT resistance selection was performed, PCR and PCR-southern testing was carried out, the phenotypic character was investigated. Artificial crossing was operated between different varieties of transgenic plants. To the F1-hybrids and their parents’inbreed offspring, ELISA test and Western blot analysis was performed. The heredity rules of il-4 gene in high generation transgenic tomatoes was revealed, the difference of il-4 protein content was analyzed. The goal is to find a varieties in which il-4 gene has a high content, and could inherit stably.In our series of experiments, 1639 seeds of T4 generation transgenic tomatoes was planted, and 1060 plants of T5 generation was obtained, among which 532 plants show PPT resistance character. There are three varieties of T5 generation plants, whose PPT resistance separation ratio matches the Mendelian rule: resistance plants: non-resistance plants = 3:1. The positive rate of il-4 gene PCR amplification is 4.15%. In the next generation, 2090 seeds of T5 generation transgenic tomatoes was planted, while 1015 plants of T6 generation was obtained, among which 485 plants show PPT resistance character. There is only one variety of T6 generation plants, whose PPT resistance separation ratio fulfills the Mendelian rule. The positive rate of il-4 gene PCR amplification after PPT selection in T6 generation is 5.32%, while the rate after non-PPT selection in T6 generation is 9.27%. In F0-hybrids, 187 seed was planted, and 117 plants of F1-hybrids was obtained. The positive rate of PCR amplification is 7.96%. The PCR-southern testing results demonstrate the existence of il-4 gene in the genome of the high generation plants. Finally, the phenotypic character of T6 generation transgenic plants and normal plants was recorded and analyzed. There is remarkable difference between T6 generation and normal plants in height, No. of fruits. Most plants of F1-hybrids have less fruits and seeds/fruit, compared to T6 generation plants. However, a few plants demonstrate heterosis. There are 44 plants of T6 generation, inbreed offspring of parents of F1-hybrids is PCR positive, among which there are 16 plants (36.36%) positive in ELISA test. The quantity of IL-4 protein is 32.49-352.51ng/FW, occupies 0.03-0.25%. In different varieties of transgenic plants, there is difference in the quantity of IL-4 protein. In the same varieties, the quantity of IL-4 protein is also different in different plants. There are 9 plants of F1-hybrids positive in PCR test, among which 8 plants (89%) is positive in ELISA test. The quantity of IL-4 protein is 26.46-643.85ng/FW, occupies 0.02-0.51%. Among three combinations of hybrids, two combinations of F1-hybrids have a higher content of IL-4 protein than their parents’inbreed offspring. Western blot analysis further confirm that IL-4 protein could inherit between generations.

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