**5. Conclusions**

Based on high-throughput sequencing, physical and chemical parameters, and statistical analysis, this study explored the effects of traffic and construction of the Qinghai-Tibet Highway on soil bacterial communities and diversity along the highway. The results showed that the road-related activities led to vegetation degradation, significantly changed the physical and chemical properties of soil, and caused heavy metal pollution. These environmental factors significantly affected the soil bacteria diversity and community structure. Soil organic carbon (SOC) and total nitrogen (TN) were the main factors driving the difference of the soil bacterial community structure in the disturbed area. The main factor of grassland disturbance along the Qinghai-Tibet Highway is caused by the decrease of soil-nutrient content. This disturbance shows a trend of increasing gradually from closer to farther distances. Therefore, in the restoration stage of the highway slope, it is very important to maintain the balance of soil nutrient supply for the restoration of the underground ecosystem function. The bacteria that showed heavy metal tolerance in the study may be explored for potential use in soil bioremediation in future studies. These results may provide guidance for the grassland ecosystem restoration along the highway and the selection of highway construction schemes in the Qinghai-Tibet region.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/1660-460 1/18/24/13137/s1, Table S1: The relative abundance of the top 10 dominant bacteria in the phylum level abundance at different altitudes and distances; Table S2: Adonis analysis in 5 m, 20 m, 50 m, 100 m and 400 m from the highway for plant and soil bacterial respectively in 3 sites and adonis analysis in altitudes of 4000 m, 4600 m and 5200 m for plant and soil bacterial.

**Author Contributions:** Conceptualization, L.H. and X.C.; methodology, Z.L.; validation, Z.L. and Y.Y.; formal analysis, Z.L.; investigation, Z.L., Y.Y., S.J., Y.L. and X.C.; resources, Z.L., S.J., D.D. and M.W.; data curation, Z.L.; writing—original draft preparation, Z.L.; writing—review and editing, L.H. and X.C.; visualization, Z.L.; supervision, L.H.; funding acquisition, X.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by [the Second Tibetan Plateau Scientific Exploration] grant number [2021QZKK0203], [Science and Technology Project of Tibet Department of Transportation] grant number [XZJTKJ[2019]01] and [National Natural Science Foundation of China] grant number [31971770].

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
