Regulation of Leaf and Reproductive organ on the Bt Protein Property in Bt cotton

摘要

Bt转基因棉的抗虫性不稳定，尤其是在棉花产量形成阶段抗虫性最低受到广泛关注。此外，在棉花所有器官中，生殖器官的抗性最低。因此我们可以推测Bt棉花抗虫性与器官发育尤其是生殖器官的发育有密切的关系。为明确Bt棉器官发育尤其是生殖器官的发育对其抗虫性的影响，通过田间和盆栽试验研究了Bt基因导入后引起棉花营养生长和生殖生长变化;氮素调节对Bt棉营养和生殖生长的影响;氨基酸组成及其浓度的变化对Bt棉杀虫蛋白含量的影响;不同密度、氮素及叶蕾调节下Bt棉蕾铃数量以及大小对其杀虫蛋白含量的影响;与器官抗虫性变化的相关的氮代谢机理等。研究结果如下:
　　1、与亲本相比，Bt棉主茎叶面积大、果枝叶片数多，株高高，果枝叶面积小;果节数、果枝数，铃数和成铃率高，果枝始节位低。N素不足和过量影响Bt棉的营养和生殖生长。与亲本相比，氮素不足导致Bt棉营养器官和生殖器官数量减少、生长速率下降。表明氮营养不足会导致Bt棉营养器官和生殖器官的生长发育下降。
　　2、相比于对照，在极端温度胁迫下，铃期比蕾期有更多的氨基酸组分及含量有更大变化，且与叶片中Bt蛋白含量的变化一致。与蕾期相比，铃期应用GPT、GS活性抑制剂MSO、AOA或者MSO和AOA共同处理时，发现有更多的氨基酸组分及更大的浓度下降，从而导致了叶片Bt蛋白含量的降低。铃期外部喷施天冬氨酸、谷氨酸、甘氨酸、脯氨酸、酪氨酸、甲硫氨酸、苯丙氨酸、组氨酸以及精氨酸，能显著增加叶片中Bt毒蛋白含量。但在蕾期只有喷施天冬氨酸、谷氨酸、脯氨酸、甲硫氨酸和精氨酸能够明显增加叶片Bt蛋白含量，且增加程度较低。因此铃期阶段Bt棉氨基酸组分及其浓度对外部应用氨基酸更敏感。此外，叶片中Bt蛋白含量与天冬氨酸、谷氨酸、甘氨酸、脯氨酸、酪氨酸、甲硫氨酸、苯丙氨酸、组氨酸、精氨酸及其浓度密切相关。
　　3、杂交种SK-3的棉蕾体积和蕾干重比常规种SK-1更大。棉蕾体积大的品种，其杀虫蛋白含量也较高。如现蕾后20天，杂交种SK-3和常规种SK-1棉蕾中杀虫蛋白含量差异均达到显著水平，且SK-3比SK-1Bt蛋白含量高5.0％。在剪叶处理下棉蕾体积和干重下降，在疏蕾处理下蕾体积和干重增加。剪叶增加了棉蕾中杀虫蛋白含量，疏蕾降低了棉蕾中杀虫蛋白含量。氨基酸、可溶性蛋白含量，GPT、GOT酶酶活性也相应地增加或下降。相反，蛋白酶和肽酶活性相应地下降或增加。在剪叶和疏蕾人工处理下，现蕾后15和30天，棉蕾大小和棉蕾中杀虫蛋白含量的呈极显著正相关（r=0.880**和r=0.720**）。
　　蕾期阶段低密度（1.50-3.00株/m2）比高密度处理（7.5株/m2）有更大的蕾体积。相应地，低密度水平下棉蕾中Bt蛋白含量也显著的高于高密度。不同种植密度下棉蕾大小和棉蕾中杀虫蛋白含量呈极显著正相关（r=0.880**）。此外，在低密度处理下，氨基酸和可溶蛋白含量、GPT和GOT活性最高，蛋白酶和肽酶活性低。N素水平也显著影响棉蕾体积及其Bt蛋白含量。施氮量增加能够显著提高棉蕾体积、棉蕾Bt蛋白含量、氨基酸和可溶蛋白含量、GPT和GOT活性、蛋白酶和肽酶活性。不同N素水平下棉蕾大小和棉蕾杀虫蛋白含量达到极显著正相关（r=0.960**）。因此，蕾体积增大有利于Bt杀虫蛋白含量提高，蕾中蛋白质合成能力增强是主要的生理原因。
　　4、种植密度显著影响Bt棉铃数、铃体积以及铃壳中Bt蛋白含量。低密度处理下，单株铃数多、铃体积大，但铃壳中Bt蛋白含量、可溶蛋白含量低，GPT和GOT活性高，氨基酸含量较高。高密度处理正相反。此外，低密度处理下铃壳蛋白酶和肽酶活性高。N素应用增加了单株铃数，铃体积，降低了铃壳中杀虫蛋白含量、可溶蛋白含量，增加了氨基酸含量、GPT和GOT活性、蛋白酶和肽酶活性。以上结果由去叶和疏蕾人工处理Bt棉株得到进一步证明。可见，促进棉铃发育降低了铃壳杀虫蛋白含量，蛋白质降解能力增强是主要的生理原因。

目录

Table of Contents

Abstract

摘要

Charter 1．General introduction

1．Overview for transgenic cotton

2．Bt cotton growth and development

2．1 Introduction of Bt gene on vegetative and reproductive organs

2．2 Introduction of Bt gene on yield and components

3．Overview for insect resistance in Bt cotcon

3．1 Bt transgenie cotton can kill bollworm significantly

3．2 Temporal and Spatial Variation in Insecticidal Efficacy in Bt Cotton

3．3 The pest could evolve resistance to Bt cotton

4．Factors affected expression of insect resistance in Bt cotton

4．1 Effects of Environmental Stress on insecticidal efficacy in Bt cotton

4．2 Mechanisms of Changes in insecticidal efficacy in Bt cotton

5．Nitrogen application on growth and insect resistance in Bt cotton

5．1 nitrogen on growth

5．2 nitrogen on insect resistance

6．Research objectives

References

Charter 2．Changed Growth Characteristics with Bacillus thuringiensis Gene Introduction and Nitrogen Regulation in Bt Cotton

1．Introduction

2 Materials and methods

2．1 Plant Material and Experimental Design

2．2 Measurements

2．3 Statistical Analysis

3 Results

3．1 Effect of the Introduction of the Bt Gene into Cotton on Vegetative Organ Growth

3．2 Effect of Introducing the Bt Gene into Cotton on Reproductive Organ Development

3．3 Effect of Nitrogen Regulation on Vegetative and Reproductive Organ Growth and Development

4 Discnssion

4．2 Effect of Nitrogen Application on Vegetative and Reproductive Growth of Bt Cotton

5 Conclusion

References

Charter 3．Amino acid Composition and Level affect Bt Protein Concentration in Bt Cotton

1．Introduction

2．Materials and methods

2．1．Plant material and experimental design

2．2 Preparation of samples

2．3 The CryIAc protein concentration assay

2．4 Determination of amino acids

2．5 Statistical analysis

3．Results

3．1 Effect of the temperature stress on leaf Bt protein and amino composition in Bt cotton

3．2 The effect of inhibitors application of GPT and GS on leaf Bt protein and amino acid composition in Bt cotton

4．Discussion

4．1 The change of amino acid metabolism by external influence related to development stage in Bt cotton

4．2 The differences in amino acid composition and their level affected Bt protein concentration in Bt cotton

5．Conclusions

References

Charter 4．Square Size affect Bt Protein Property in Bt Cotton

1．Introduction

2．Materials and methods

2．1．Plant materials and field design

2．2．Preparation of plant material

2．3．Physiological measurements

2．4．Statistics analysis

3．Results

3．1 square size on insecticidal property for different type of cultivars

3．2 Square size on insecticidal property under different planting density for the cultivars

3．3 Square size on insecticidal property under different nitrogen rates for the cultivars

3．5．Square size on insecticidal property by leaf-square regulation for the cultivars

4．Discussion

4．2．Big square formation in breeding and cultural practices benefit to increase insect resistance

Referenees

Charter 5．Boll Number and Size on Boll Wall Insecticidal Protein Content in Bt Cotton

1．Introduction

2．Materials and Methods

2．1．Plant materials and field design

2．2．Preparation of plant material

2．3．Physiological measurements

2．4．Statistics analysis

3．Results

3．1 boll number and size on insecticidal property under different planting density for the cultivars

3．2 boll number and size on insecticidal property under different nitrogen rates for the cultivars

3．3．boll number，size on boll wall insecticidal property by leaf-square regulation for the cultivars

4．Discussions

5．Conclusion

References

Acknowledgment

The publications of the author

声明